Ester prodrugs of prostratin and related phorbol compounds

ABSTRACT

The present disclosure provides analogs and derivatives of phorbol compounds for the treatment of viral infections, neoplastic diseases, inflammatory reactions, and use as analgesics.

1. CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.11/995,527, filed Jan. 11, 2008, which is a National Stage applicationunder 35 U.S.C. §371 of International Application No. PCT/US2006/027303,filed Jul. 13, 2006, which claims benefit under 35 U.S.C. §119(e) ofU.S. provisional application Ser. No. 60/699,047, filed Jul. 13, 2005,the contents of each of which are incorporated herein by reference intheir entirety.

2. TECHNICAL FIELD

The present disclosure relates to analogs and derivatives of phorbolcompounds, their synthesis, and methods of using the compounds.

3. BACKGROUND

While highly active antiretroviral therapy (HAART) is helping manypeople with HIV/AIDS live longer, healthier lives, these combination or“cocktail” treatments are not always effective and often cause seriousadverse side effects. Even among those who do well on HAART, manypatients experience treatment failure within a year or two, oftenbecause HIV has developed resistance to the drugs used to inhibit itsgrowth. Drug resistant HIV tends to progressively develop resistance toother drugs in the same class, or even to the entire class of drugs. Inaddition, many people newly infected with HIV may carry viral strainsalready resistant to current treatments.

Confounding the resistance challenge is the latency of HIV infection.Despite the success of HAART, medical reports suggest thatantiretroviral therapy alone is unable to eliminate the viral infectionbecause the virus can persist in a latent form when infected CD4⁺lymphoblasts carrying an integrated copy of the HIV-1 genome revert backto a resting memory state. In this state, CD4 cells are minimallypermissive for virus gene expression, and infected memory cells cansurvive for many years. Following reexposure to the relevant antigen orother activating stimuli, these cells can begin to produce virus again.Though HAART may be effective for an acute infection, it appears unableto eliminate this viral reservoir, which can serve as a permanentarchive for wild-type virus and for drug-resistant variants that ariseduring treatment. Thus, once resistance to a particular drug arises, thepatient may always carry that resistant viral strain (Siliciano et al.,2004, J. Antimicrobial Chemo. 54:6-9).

Commonly used anti-HIV therapies generally target two viral enzymes thatHIV needs to reproduce: reverse transcriptase and protease. A thirdtherapeutic approach is to target the binding of the virus to the cell,thereby affecting the viral entry phase of the HIV replication cycle. Anexample of the latter therapy is enfurvirtide (Fuzeon®), a linear 36amino acid peptide that binds to the heptad repeat in the gp41 viralenvelope glycoprotein on CD4⁺ cells. However, because of the problemsassociated with drug resistance and virus latency, there is a need fornew therapeutic approaches based on anti-viral agents that arestructurally and/or mechanistically different from the currentlyapproved compounds. One such approach is represented by the anti-viralproperties of phorbol compounds prostratin (see, e.g., U.S. Pat. No.5,599,839) and ingenol (see, e.g., Fujiwara et al., 1996, AntimicrobAgents Chemother. 40(1):271-3).

Prostratin, 12-deoxyphorbol 13-acetate, was first isolated from Pimeleaprostrata, a New Zealand plant toxic to livestock (Zayed S., 1977,Experentia 33(12):1554-5). Prostratin was subsequently isolated fromHomalanthus nutans, a medicinal plant used by traditional Samoanhealers, and shown to inhibit HIV-induced cell killing and viralreplication in a variety of cell systems (Cox et al, 1993, J.Ethnopharmacol. 38(2-3):181-8; Cox, P. A., 1994, Ciba Found. Symp.185:25-36). The potency and degree of cytoprotection is dependent onboth viral strain and host cell type (Gustafson et al, 1992, J. Med.Chem. 35(11):1978-86).

Similarly, ingenol compounds were used in traditional medicine for thetreatment of skin conditions (e.g., warts, corns, etc.), cancer, andasthma (Green et al., 1988, Australian J Dermatol 29:127-30 and Weedonet al., 1976, Med J of Australia 1:928). Ingenol-3,5,20-triacetate hasalso been show to have anti-viral properties (Fujiwara et al., supra).

Interestingly, studies suggest that prostratin displays a unique dualeffect on HIV biology inhibiting HIV replication while activatingdormant or “latent” HIV that hides in human cells (Kulkosky et al.,2001, Blood. 98(10):3006-15). Prostratin efficiently reactivates HIVexpression from latently infected cells generated in a SCID-hu mouse.Reactivation is associated with induction of viral transcription fromthe HIV long terminal repeat (LTR) and is thought to involveprostratin's property of activating specific protein kinase C (PKC)isozymes. Prostratin also appears to inhibit the entry step of the HIVreplication cycle by interacting with a cellular target necessary forviral entry and/or by downregulating HIV co-receptors CCR5 and CXR5(Witvrouw et al, 2003, Antivir Chem. Chemother. 14(6):321-8).Prostratin's unique mechanism of action is indicated by itseffectiveness against different strains of HIV-1, such as HIV subtypes Band D, clinical HIV isolate (L1), HIV-2 (ROD and EHO), and SIV (MA C251) and effectiveness against HIV strains resistant to polyanionicbinding inhibitor dextran sulfate, the fusion inhibitor enfuvirtride,nucleoside reverse transcriptase inhibitors (NRTs), and proteaseinhibitor (PIs) (Witvrouw et al., supra). Likewise, ingenols withantiviral activity appear to affect the viral absorption process ratherthan the viral replication machinery (Fugiwara et al., supra). Studiesfurther suggest that ingenol compounds may also cause reactivation oflatent viruses, similar to the effects seen with prostratin (Fujiwara etal., 1998, Arch Virol. 143(10):2003-10).

Although prostratin and related phorbol compounds present an attractivetherapeutic strategy for HIV in view of their reactivation and antiviralproperties, as well as treatments for other diseases affected throughphorbol mediated signal transduction pathways, the therapeuticeffectiveness of these phorbol compounds is limited by their lowsolubility, low oral bioavailability, and low therapeutic index.

4. SUMMARY

The present disclosure provides analogs and derivatives of prostratinand related phorbol compounds, methods of making the compounds, andmethods of using the compounds to treat various conditions and diseases.The phorbol compounds disclosed herein are prodrugs of phorbol estersthat have various biological activities, including binding to phorbolreceptors and modulation of associated signal transduction pathways,reactivation of latent viruses and inhibition of viral absorption, andtumor promoting properties as well as tumor promoter inhibitingactivity. In relation to these biological effects, the prodrugs finduses in treating pain, cell proliferative disorders, inflammatoryreactions, and viral infections.

In some aspects, the compounds disclosed herein are prodrug compoundsaccording to structural formula (I):R—X—O—C(O)—R′  (I)

including salts and hydrates thereof, wherein:

R is a residue of a phorbol ester;

X is an alkylene chain containing from 1 to 12 carbon atoms;

R′ is a moiety that either bears a permanent charge or that is ionizableat a pH in the range of about 2 to about 8;

the illustrated —X—O—C(O)—R′ group is linked to the 6-carbon of R; and

the illustrated —X—O—C(O)—R′ group hydrolyzes under biologicalconditions to yield a group of the formula —X—OH.

In some embodiments of the compound of structural formula (I), the X ismethano (—CH₂—) and R′ is a group that comprises a carboxyl group or asalt thereof.

In some embodiments, the R′ is a group of the formula —(CH₂)_(m)—C(O)OM,where M is hydrogen or a counter ion and m is an integer ranging from 1to 4.

In some embodiments, the R′ can be selected from—(CH₂)_(n)CH[(CH₂)_(n)—NH₂]—(CH₂)_(n)—C(O)OM and —(CH₂)_(n)—CH(NH₂)_(n)(CH₂)_(n)—C(O)OM, where M is as defined as above and each n is,independently of the others, an integer ranging from 0 to 4.

In some embodiments, the R′ of the phorbol compounds of structuralformula (I) is a group of the formula —Y—Z, wherein:

Y is a branched or unbranched, saturated or unsaturated alkylene chaincontaining from 1 to 4 carbon atoms;

Z is selected from —C(O)OM, —NR^(b)R^(b) and —NR^(c)R^(c)R^(c);

M is hydrogen or a counter ion;

each R^(b) is, independently of the other, selected from hydrogen, loweralkyl, lower hydroxyalkyl and lower alkoxyalkyl, heteroalkyl or,alternatively, two R^(b) groups bonded to the same nitrogen atom may betaken together with the nitrogen atom to which they are bonded to form a5- to 7-membered heteroatomic ring; and

each R^(c) is, independently of the others, selected from lower alkyl,lower hydroxyalkyl and lower alkoxyalkyl.

In some embodiments, R is selected from structures (R1) and (R2):

wherein:

R″ is selected from (C₁-C₁₄) alkyl and benzyl.

In other aspects, provided herein are pharmaceutical compositions of theprodrug compounds, or pharmaceutically acceptable salts and hydratesthereof, and a pharmaceutically acceptable vehicle, such as anexcipient, diluent or carrier. The choice of vehicle will depend upon,among other factors, the mode of administration.

In some aspects, the prodrug compounds can be used to modulate theactivity of a phorbol receptor, or a signal transduction cascadedependent thereon. Generally, the method comprises administering to acell the prodrug compound under conditions in which the progroup cleavesto generate an active phorbol compound.

In other aspects, the prodrug compounds can be used to treat variousconditions or diseases, such as neoplasms, viral infections,inflammatory conditions, and pain. The methods comprise administering toa subject afflicted with the condition or disease an amount of theprodrug compound effective to treat the condition or disease. Thecompounds can also be used as prophylaxis in reducing the risk of theoccurrence of the condition or disease.

Further provided herein are kits comprising the prodrug compounds. Thecompounds can be provided as pharmaceutical compositions in variousdosage forms and dosage units for administration.

5. DETAILED DESCRIPTION 5.1 Definitions

As used throughout the instant application, the following terms shallhave the following meanings:

“Alkyl” by itself or as part of another substituent refers to asaturated or unsaturated branched, straight-chain or cyclic monovalenthydrocarbon radical having the stated number of carbon atoms (i.e.,C₁-C₆ means one to six carbon atoms) that is derived by the removal ofone hydrogen atom from a single carbon atom of a parent alkane, alkeneor alkyne. Typical alkyl groups include, but are not limited to, methyl;ethyls such as ethanyl, ethenyl, ethynyl; propyls such as propan-1-yl,propan-2-yl, cyclopropan-1-yl, prop-1-en-1-yl, prop-1-en-2-yl,prop-2-en-1-yl, cycloprop-1-en-1-yl; cycloprop-2-en-1-yl,prop-1-yn-1-yl, prop-2-yn-1-yl, etc.; butyls such as butan-1-yl,butan-2-yl, 2-methyl-propan-1-yl, 2-methyl-propan-2-yl, cyclobutan-1-yl,but-1-en-1-yl, but-1-en-2-yl, 2-methyl-prop-1-en-1-yl, but-2-en-1-yl,but-2-en-2-yl, buta-1,3-dien-1-yl, buta-1,3-dien-2-yl,cyclobut-1-en-1-yl, cyclobut-1-en-3-yl, cyclobuta-1,3-dien-1-yl,but-1-yn-1-yl, but-1-yn-3-yl, but-3-yn-1-yl, etc.; and the like.

The term “alkyl” is specifically intended to include groups having anydegree of level of saturation, e.g., groups having exclusively singlecarbon-carbon bonds, groups having one or more double carbon bonds,groups having mixtures of single, double, and triple carbon-carbonbonds. Where specific levels of saturation are intended, thenomenclature “alkanyl,” “alkenyl” and/or “alkynyl” is used, as definedbelow. In some embodiments, the alkyl group comprises from 1 to 20carbon atoms (C₁-C₂₀). In some embodiments, the alkyl group comprises 1to 10 carbon atoms (C₁-C₁₀). In some embodiments, an alkyl groupcomprises from 1 to 6 carbon atoms (C₁-C₆). The expression “lower alkyl”refers to alkyl groups comprised of from 1 to 6 carbon atoms.

“Alkanyl” by itself or as part of another substituent refers to asaturated branched, straight-chain or cyclic alkyl derived by theremoval of one hydrogen atom from a single carbon atom of a parentalkane. Typical alkanyl groups include, but are not limited to,methanyl; ethanyl; propanyls such as propan-1-yl, propan-2-yl(isopropyl), cyclopropan-1-yl, etc.; butanyls such as butan-1-yl,butan-2-yl (sec-butyl), 2-methyl-propan-1-yl (isobutyl),2-methyl-propan-2-yl (t-butyl), cyclobutan-1-yl, etc; and the like.

“Alkenyl” by itself or as part of another substituent refers to anunsaturated branched, straight-chain or cyclic alkyl having at least onecarbon-carbon double bond derived by the removal of one hydrogen atomfrom a single carbon atom of a parent alkene. The group may be in eitherthe cis or trans conformation about the double bond(s). Typical alkenylgroups include, but are not limited to, ethenyl; propenyls such asprop-1-en-1-yl, prop-1-en-2-yl, prop-2-en-1-yl, prop-2-en-2-yl,cycloprop-1-en-1-yl; cycloprop-2-en-1-yl; butenyls such asbut-1-en-1-yl, but-1-en-2-yl, 2-methyl-prop-1-en-1-yl, but-2-en-1-yl,but-2-en-2-yl, buta-1,3-dien-1-yl, buta-1,3-dien-2-yl,cyclobut-1-en-1-yl, cyclobut-1-en-3-yl, cyclobuta-1,3-dien-1-yl, etc;and the like. In some embodiments, the alkenyl group is (C₂-C₆) alkenyl.

“Alkenyl” by itself or as part of another substituent refers to anunsaturated branched, straight-chain or cyclic alkyl having at least onecarbon-carbon triple bond derived by the removal of one hydrogen atomfrom a single carbon atom of a parent alkyne. Typical alkynyl groupsinclude, but are not limited to, ethynyl; propynyls such asprop-1-yn-1-yl, prop-2-yn-1-yl, etc.; butynyls such as but-1-yn-1-yl,but-1-yn-3-yl, but-3-yn-1-yl, etc; and the like. In some embodiments,the alkynyl group is (C₂-C₆) alkynyl.

“Alkyldiyl” by itself or as part of another substituent refers to asaturated or unsaturated, branched, straight-chain or cyclic divalenthydrocarbon group having the stated number of carbon atoms (e.g., C₁-C₆means from one to six carbon atoms) derived by the removal of onehydrogen atom from each of two different carbon atoms of a parentalkane, alkene or alkyne, or by the removal of two hydrogen atoms from asingle carbon atom of a parent alkane, alkene or alkyne. The twomonovalent radical centers or each valency of the divalent radicalcenter can form bonds with the same or different atoms. Typicalalkyldiyl groups include, but are not limited to, methandiyl; ethyldiylssuch as ethan-1,1-diyl, ethan-1,2-diyl, ethen-1,1-diyl, ethen-1,2-diyl;propyldiyls such as propan-1,1-diyl, propan-1,2-diyl, propan-2,2-diyl,propan-1,3-diyl, cyclopropan-1,1-diyl, cyclopropan-1,2-diyl,prop-1-en-1,1-diyl, prop-1-en-1,2-diyl, prop-2-en-1,2-diyl,prop-1-en-1,3-diyl, cycloprop-1-en-1,2-diyl, cycloprop-2-en-1,2-diyl,cycloprop-2-en-1,1-diyl, prop-1-yn-1,3-diyl, etc.; butyldiyls such as,butan-1,1-diyl, butan-1,2-diyl, butan-1,3-diyl, butan-1,4-diyl,butan-2,2-diyl, 2-methyl-propan-1,1-diyl, 2-methyl-propan-1,2-diyl,cyclobutan-1,1-diyl; cyclobutan-1,2-diyl, cyclobutan-1,3-diyl,but-1-en-1,1-diyl, but-1-en-1,2-diyl, but-1-en-1,3-diyl,but-1-en-1,4-diyl, 2-methyl-prop-1-en-1,1-diyl,2-methanylidene-propan-1,1-diyl, buta-1,3-dien-1,1-diyl,buta-1,3-dien-1,2-diyl, buta-1,3-dien-1,3-diyl, buta-1,3-dien-1,4-diyl,cyclobut-1-en-1,2-diyl, cyclobut-1-en-1,3-diyl, cyclobut-2-en-1,2-diyl,cyclobuta-1,3-dien-1,2-diyl, cyclobuta-1,3-dien-1,3-diyl,but-1-yn-1,3-diyl, but-1-yn-1,4-diyl, buta-1,3-diyn-1,4-diyl, etc.; andthe like. Where specific levels of saturation are intended, thenomenclature alkanyldiyl, alkenyldiyl and/or alkynyldiyl is used. Whereit is specifically intended that the two valencies are on the samecarbon atom, the nomenclature “alkylidene” is used. In some embodiments,the alkyldiyl group is (C₁-C₆) alkyldiyl. In some embodiments, thealkyldiyl groups are saturated acyclic alkanyldiyl groups in which theradical centers are at the terminal carbons, e.g., methandiyl (methano);ethan-1,2-diyl (ethano); propan-1,3-diyl (propano); butan-1,4-diyl(butano); and the like (also referred to as alkylenos, defined infra).

“Alkyleno” by itself or as part of another substituent refers to astraight-chain saturated or unsaturated alkyldiyl group having twoterminal monovalent radical centers derived by the removal of onehydrogen atom from each of the two terminal carbon atoms ofstraight-chain parent alkane, alkene or alkyne. The locant of a doublebond or triple bond, if present, in a particular alkyleno is indicatedin square brackets. Typical alkyleno groups include, but are not limitedto, methano; ethylenos such as ethano, etheno, ethyno; propylenos suchas propano, prop[1]eno, propa[1,2]dieno, prop[1]yno, etc.; butylenossuch as butano, but[1]eno, but[2]eno, buta[1,3]dieno, but[1]yno,but[2]yno, buta[1,3]diyno, etc.; and the like. Where specific levels ofsaturation are intended, the nomenclature alkano, alkeno and/or alkynois used. In some embodiments, the alkyleno group is (C₁-C₆) alkyleno. Insome embodiments, the alkyleno group is (C₁-C₃) alkyleno. In someembodiments, the alkyleno groups are straight-chain saturated alkanogroups, e.g., methano, ethano, propano, butano, and the like.

“Acyl” by itself or as part of another substituent refers to —C(O)R⁵,where R⁵ is hydrogen, or substituted or unsubstituted alkyl, cylcoalkyl,cycloheteroalkyl, aryl, arylalkyl, heteroalkyl, heteroaryl, orheteroarylalkyl as defined herein. Typical acyl groups include, but arenot limited to, formyl, acetyl, cyclohexylcarbonyl,cyclohexylmethylcarbonyl, benzoyl, benzylcarbonyl, and the like.

“Acyloxy” by itself or as part of another substituent refers to—OC(O)R⁶, where R⁶ represents a hydrogen, or substituted orunsubstituted alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl,arylalkyl, and heteroaryl groups as defined herein. Alkylacyloxy refersan acyloxy where R⁶ is (C₁-C₁₂) alkyl, (C₁-C₈) alkyl, or (C₁-C₄) alkyl.Arylacyloxy refers to an acyloxy where R⁶ is an aryl optionallysubstituted with selected substituents, including, but not limited to,hydroxyl, alkyl, halogen, (C₁-C₄) alkyl, (C₁-C₄) alkoxy, and carboxyl.

“Alkoxy” by itself or as part of another substituent refers to —OR′,where R⁷ represents an alkyl or cycloalkyl group as defined herein.Typical alkoxy groups include, but are not limited to, methoxy, ethoxy,propoxy, butoxy, cyclohexyloxy, and the like.

“Alkoxycarbonyl” by itself or as part of another substituent, refers to—C(O)OR⁸ where R⁸ represents an alkyl or cyclalkyl group as definedherein. Typical alkoxycarbonyl groups include, but are not limited to,methoxycarbonyl, ethoxycarbonyl, proproxycarbonyl, butoxycarbonyl,cyclohexyloxycarbonyl, and the like.

“Amino” by itself or as part of another substituent refers to the group—NH₂. Substituted amino refers to the group —NHR⁹, NR⁹R⁹, and NR⁹R⁹R⁹,where each R⁹ is independently selected from substituted orunsubstituted alkyl, cycloalkyl, cycloheteroalkyl, alkoxy, aryl,heteroaryl, heteroarylalkyl, acyl, alkoxycarbonyl, sulfanyl, sulfinyl,sulfonyl, and the like. Typical amino groups include, but are limitedto, dimethylamino, diethylamino, trimethylamino, triethylamino,methylysulfonylamino, furanyl-oxy-sulfamino, and the like.

“Heteroalkyl,” Heteroalkanyl,” Heteroalkenyl,” Heteroalkynyl,”Heteroalkyldiyl” and “Heteroalkyleno” by themselves or as part ofanother substituent refer to alkyl, alkanyl, alkenyl, alkynyl, alkyldiyland alkyleno groups, respectively, in which one or more of the carbonatoms are each independently replaced with the same or differentheteratoms or heteroatomic groups. Typical heteroatoms and/orheteroatomic groups which can replace the carbon atoms include, but arenot limited to, —O—, —S—, —S—O—, —NR¹⁰—, —PR¹⁰—, —S(O)—, —S(O)₂—,—S(O)NR¹⁰—, —S(O)₂NR¹⁰—, etc., including combinations thereof, whereeach R¹⁰ is independently hydrogen, or substituted or unsubstitutedalkyl, aryl, arylalkyl, cycloalkyl, cycloheteroalkyl heteroalkyl,heteroaryl, or heterarylalkyl.

“Cycloalkyl” by itself or as part of another substituent refers to asaturated or unsaturated cyclic alkyl derived by removal of one hydrogenfrom the parent cyclic alkyl. Where a specific level of saturation isintended, the nomemclature “cycloalkanyl” or “cycloalkenyl” is used.Typical cycloalkyl groups include, but are not limited to, cyclopropyl;cyclobutyls such as cyclobutanyl and cyclobutenyl; cyclopentyls such ascyclopentanyl and cyclopentenyl; cyclohexyls such as cyclohexanyl andcyclohexenyl; and the like. In some embodiments, the cycloalkyl group is(C₃-C₁₀) cycloalkyl. In other embodiments, the cycloalkyl group is(C₃-C₇) cycloalkyl.

“Cycloheteroalkyl” by itself or as part of another substituent refers toa saturated or unsaturated cyclic alkyl in which one or more carbonatoms (and any associated hydrogen atoms) are independently replacedwith the same or different heteroatom. Typical heteroatoms to replacethe carbon atom include but are not limited to N, P, O, S, etc. Aheteroatom can occupy the position that is attached to the remainder ofthe molecule. Typical heterocycloalkyl groups include, but are notlimited to, tetrahydrofuranyl (e.g., tetrahydrofuran-2-yl,tetrahydrofuran-3-yl, etc.), piperidinyl (e.g., piperidin-1-yl,piperidin-2-yl, etc.), morpholinyl (e.g., morpholin-3-yl,morpholin-4-yl, etc.), piperazinyl (e.g., piperazin-1-yl,piperazin-2-yl, etc.), pyrrolidinyl, etc.; and the like.

“Parent Aromatic Ring System” refers to an unsaturated cyclic orpolycyclic ring system having a conjugated electron system. Specificallyincluded within the definition of “parent aromatic ring system” arefused ring systems in which one or more of the rings are aromatic andone or more of the rings are saturated or unsaturated, such as, forexample, fluorene, indane, indene, phenalene, tetrahydronaphthalene,etc. Typical parent aromatic ring systems include, but are not limitedto, aceanthrylene, acenaphthylene, acephenanthrylene, anthracene,azulene, benzene, chrysene, coronene, fluoranthene, fluorene, hexacene,hexaphene, hexylene, indacene, s-indacene, indane, indene, naphthalene,octacene, octaphene, octalene, ovalene, penta-2,4-diene, pentacene,pentalene, pentaphene, perylene, phenalene, phenanthrene, picene,pleiadene, pyrene, pyranthrene, rubicene, tetrahydronaphthalene,triphenylene, trinaphthalene, etc., as well as the various hydro isomersthereof.

“Aryl” by itself or as part of another substituent refers to amonovalent aromatic hydrocarbon group having the stated number of carbonatoms (i.e., C₅-C₁₅ means from 5 to 15 carbon atoms) derived by theremoval of one hydrogen atom from a single carbon atom of a parentaromatic ring system. Typical aryl groups include, but are not limitedto, groups derived from aceanthrylene, acenaphthylene,acephenanthrylene, anthracene, azulene, benzene, chrysene, coronene,fluoranthene, fluorene, hexacene, hexaphene, hexylene, as-indacene,s-indacene, indane, indene, naphthalene, octacene, octaphene, octalene,ovalene, penta-2,4-diene, pentacene, pentalene, pentaphene, perylene,phenalene, phenanthrene, picene, pleiadene, pyrene, pyranthrene,rubicene, triphenylene, trinaphthalene, and the like, as well as thevarious hydro isomers thereof. In some embodiments, the aryl groupcomprises (C₅-C₁₅) aryl. In other embodiments, the aryl group comprises(C₅-C₁₀) aryl.

“Arylalkyl” by itself or as part of another substituent refers to anacyclic alkyl group in which one of the hydrogen atoms bonded to acarbon atom, typically a terminal or sp³ carbon atom, is replaced withan aryl group. Typical arylalkyl groups include, but are not limited to,benzyl, 2-phenylethan-1-yl, 2-phenylethen-1-yl, naphthylmethyl,2-naphthylethan-1-yl, 2-naphthylethen-1-yl, naphthobenzyl,2-naphthophenylethan-1-yl and the like. Where specific alkyl moietiesare intended, the nomenclature arylalkanyl, arylakenyl and/orarylalkynyl is used. In some embodiments, the arylalkyl group is(C₆-C₂₁) arylalkyl, e.g., the alkanyl, alkenyl or alkynyl moiety of thearylalkyl group is (C₁-C₆) and the aryl moiety is (C₅-C₁₅). In otherembodiments the arylalkyl group is (C₆-C₁₃), e.g., the alkanyl, alkenylor alkynyl moiety of the arylalkyl group is (C₁-C₃) and the aryl moietyis (C₅-C₁₀)—

“Aryloxy” by itself or as part of another substituent, refers to aradical of the formula —O-aryl, where aryl is as defined herein.

“Arylalkyloxy” by itself or as part of another substituent, refers to aradical of the formula —O-arylalkyl, where arylalkyl is as definedherein.

“Aryloxycarbonyl” by itself or as part of another substituent, refers toa radical of the formula —C(O)—O-aryl, where aryl is as defined herein.

“Parent Heteroaromatic Ring System” refers to a parent aromatic ringsystem in which one or more carbon atoms are each independently replacedwith the same or different heteroatoms or heteroatomic groups. Typicalheteroatoms or heteroatomic groups to replace the carbon atoms include,but are not limited to, N, NH, P, O, S, S(O), S(O)₂, Si, etc.Specifically included within the definition of “parent heteroaromaticring systems” are fused ring systems in which one or more of the ringsare aromatic and one or more of the rings are saturated or unsaturated,such as, for example, benzodioxan, benzofuran, chromane, chromene,indole, indoline, xanthene, etc. Also included in the definition of“parent heteroaromatic ring system” are those recognized rings thatinclude common substituents, such as, for example, benzopyrone and1-methyl-1,2,3,4-tetrazole. Specifically excluded from the definition of“parent heteroaromatic ring system” are benzene rings fused to cyclicpolyalkylene glycols such as cyclic polyethylene glycols. Typical parentheteroaromatic ring systems include, but are not limited to, acridine,benzimidazole, benzisoxazole, benzodioxan, benzodioxole, benzofuran,benzopyrone, benzothiadiazole, benzothiazole, benzotriazole,benzoxaxine, benzoxazole, benzoxazoline, carbazole, -carboline,chromane, chromene, cinnoline, furan, imidazole, indazole, indole,indoline, indolizine, isobenzofuran, isochromene, isoindole,isoindoline, isoquinoline, isothiazole, isoxazole, naphthyridine,oxadiazole, oxazole, perimidine, phenanthridine, phenanthroline,phenazine, phthalazine, pteridine, purine, pyran, pyrazine, pyrazole,pyridazine, pyridine, pyrimidine, pyrrole, pyrrolizine, quinazoline,quinoline, quinolizine, quinoxaline, tetrazole, thiadiazole, thiazole,thiophene, triazole, xanthene, and the like.

“Heteroaryl” by itself or as part of another substituent refers to amonovalent heteroaromatic group having the stated number of ring atoms(e.g., “5-14 membered” means from 5 to 14 ring atoms) derived by theremoval of one hydrogen atom from a single atom of a parentheteroaromatic ring system. Typical heteroaryl groups include, but arenot limited to, groups derived from acridine, benzimidazole,benzisoxazole, benzodioxan, benzodiaxole, benzofuran, benzopyrone,benzothiadiazole, benzothiazole, benzotriazole, benzoxazine,benzoxazole, benzoxazoline, carbazole, -carboline, chromane, chromene,cinnoline, furan, imidazole, indazole, indole, indoline, indolizine,isobenzofuran, isochromene, isoindole, isoindoline, isoquinoline,isothiazole, isoxazole, naphthyridine, oxadiazole, oxazole, perimidine,phenanthridine, phenanthroline, phenazine, phthalazine, pteridine,purine, pyran, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine,pyrrole, pyrrolizine, quinazoline, quinoline, quinolizine, quinoxaline,tetrazole, thiadiazole, thiazole, thiophene, triazole, xanthene, and thelike, as well as the various hydro isomers thereof. In some embodiments,the heteroaryl group is a 5-20 membered heteroaryl. In otherembodiments, the heteroaryl group is a 5-10 membered heteroaryl.

“Heteroarylalkyl” by itself or as part of another substituent refers toan acyclic alkyl group in which one of the hydrogen atoms bonded to acarbon atom, typically a terminal or sp³ carbon atom, is replaced with aheteroaryl group. Where specific alkyl moieties are intended, thenomenclature heteroarylalkanyl, heteroarylalkenyl and/orheteroarylalkynyl is used. In some embodiments, the heteroarylalkylgroup is a 6-20 membered heteroarylalkyl, e.g., the alkanyl, alkenyl oralkynyl moiety of the heteroarylalkyl is (C₁-C₁₀) alkyl and theheteroaryl moiety is a 5-12-membered heteroaryl. In some embodiments,the heteroarylalkyl is a 6-13 member heteroarylalkyl, e.g., the alkanyl,alkenyl or alkynyl moiety of the heteroarylalkyl is (C₁-C₃) alkyl andthe heteroaryl moiety is a 5-10 membered heteroaryl.

“Halogen” or “Halo” by themselves or as part of another substituent,unless otherwise stated, refer to fluoro, chloro, bromo and iodo.

“Haloalkyl” by itself or as part of another substituent refers to analkyl group in which one or more of the hydrogen atoms is replaced witha halogen. Thus, the term “haloalkyl” is meant to includemonohaloalkyls, dihaloalkyls, trihaloalkyls, etc. up to perhaloalkyls.For example, the expression “(C₁-C₂) haloalkyl” includes fluoromethyl,difluoromethyl, trifluoromethyl, 1-fluoroethyl, 1,1-difluoroethyl,1,2-difluoroethyl, 1,1,1-trifluoroethyl, perfluoroethyl, etc.

The above-defined groups may include prefixes and/or suffixes that arecommonly used in the art to create additional well-recognizedsubstituent groups. As examples, “alkyloxy” or “alkoxy” refers to agroup of the formula —OR⁷, as defined above; “alkylamine” refers to agroup of the formula —NHR¹¹; and “dialkylamine” refers to a group of theformula —NR¹¹R¹¹, where each R¹¹ is independently an alkyl. As anotherexample, “haloalkoxy” or “haloalkyloxy” refers to a group of the formula—OR¹², where R¹² is a haloalkyl.

“Substituted” when used to modify a specified group or radical, meansthat one or more hydrogen atoms of the specified group or radical areeach, independently of one another, replaced with the same or differentsubstituent(s). Substituent groups useful for substituting saturatedcarbon atoms in the specified group or radical include, but are notlimited to —R¹³, halo, —O⁻, ═O, —OR¹⁴, —SR¹⁴, —S⁻, ═S, —NR¹⁵NR¹⁵, ═N¹⁴,═N—OR¹⁴, trihalomethyl, —CF₃, —CN, —OCN, —SCN, —NO, —NO₂, ═N₂, —N₃,—S(O)₂R¹⁴, —S(O)₂O⁻, —S(O)₂OR¹⁴, —OS(O)₂R¹⁴, —OS(O)₂O⁻, —OS(O)₂OR¹⁴,—P(O)(O⁻)₂, —P(O)(OR¹⁴)(O⁻), —P(O)(OR¹⁴)(OR¹⁴), —C(O)R¹⁴, —C(S)R¹⁴,—C(NR¹⁴)R¹⁴, —C(O)O⁻, —C(O)OR¹⁴, —C(S)OR¹⁴, —C(O)NR¹⁵R¹⁵,—C(NR¹⁴)NR¹⁵R¹⁵, —OC(O)R¹⁴, —OC(S)R¹⁴, —OC(O)O⁻, —OC(O)OR¹⁴, —OC(S)OR¹⁴,—NR¹⁴C(O)R¹⁴, —NR¹⁴C(S)R¹⁴, —NR¹⁴C(O)O⁻, —NR¹⁴C(O)OR¹⁴, —NR¹⁴C(S)OR¹⁴,—NR¹⁴C(O)NR¹⁵R¹⁵, —NR¹⁴C(NR¹⁴)R¹⁴ and —NR¹⁴C(NR¹⁴)NR¹⁵R¹⁵, where R¹³ isselected from the group consisting of alkyl, cycloalkyl, heteroalkyl,cycloheteroalkyl, aryl, arylalkyl, heteroaryl and heteroarylalkyl; eachR¹⁴ is independently hydrogen or R¹³; and each R¹⁵ is independently R¹⁴or alternatively, the two R¹⁵s are taken together with the nitrogen atomto which they are bonded form a 5-, 6- or 7-membered cycloheteroalkylwhich may optionally include from 1 to 4 of the same or differentadditional heteroatoms selected from the group consisting of O, N and S.As specific examples, —NR¹⁵R¹⁵ is meant to include —NH₂, —NH-alkyl,N-pyrrolidinyl and N-morpholinyl.

Similarly, substituent groups useful for substituting unsaturated carbonatoms in the specified group or radical include, but are not limited to,—R¹³, halo, —O⁻, —OR¹⁴, —SR¹⁴, —S⁻, —NR¹⁵R¹⁵, trihalomethyl, —CF₃, —CN,—OCN, —SCN, —NO, —NO₂, —N₃, —S(O)₂R¹⁴, —S(O)₂O⁻, —S(O)₂OR¹⁴, —OS(O)₂R¹⁴,—OS(O)₂OR⁻, —OS(O)₂OR¹⁴, —P(O)(O⁻)₂, —P(O)(OR¹⁴)(O⁻), —P(O)(OR¹⁴)(OR¹⁴),—C(O)R¹⁴, —C(S)R¹⁴, —C(NR¹⁴)R¹⁴, —C(O)O⁻, —C(O)OR¹⁴, —C(S)OR¹⁴,—C(O)NR¹⁵R¹⁵, —C(NR¹⁴)NR¹⁵R¹⁵, —OC(O)R¹⁴, —OC(S)R¹⁴, —OC(O)O⁻,—OC(O)OR¹⁴, —OC(S)OR¹⁴, —NR¹⁴C(O)R¹⁴, —NR¹⁴C(S)R¹⁴, —NR¹⁴C(O)O⁻,—NR¹⁴C(O)OR¹⁴, NR¹⁴C(S)OR¹⁴, —NR¹⁴C(O)NR¹⁵R¹⁵, —R¹⁴C(NR¹⁴)R¹⁴ and—NR¹⁴C(NR¹⁴)NR¹⁵R¹⁵, where R¹³, R¹⁴ and R¹⁵ are as previously defined.

Substituent groups useful for substituting nitrogen atoms in heteroalkyland cycloheteroalkyl groups include, but are not limited to, —R¹³, —O⁻,—OR¹⁴, —SR¹⁴, —S⁻, —NR¹⁵R¹⁵, trihalomethyl, —CF₃, —CN, —NO, —NO₂,—S(O)₂R¹⁴, —S(O)₂O⁻, —S(O)₂OR¹⁴, —OS(O)₂R¹⁴, —OS(O)₂O⁻, —OS(O)₂OR¹⁴,—P(O)(O⁻)₂, —P(O)(OR¹⁴)(O⁻), —P(O)(OR¹⁴)(OR¹⁴), —C(O)R¹⁴, —C(S)R¹⁴,—C(NR¹⁴)R¹⁴, —C(O)OR¹⁴, —C(S)OR¹⁴, —C(O)NR¹⁵R¹⁵, —C(NR¹⁵)NR¹⁵R¹⁵,—OC(O)R¹⁴, —OC(S)R¹⁴, —OC(O)OR¹⁴, —OC(S)OR¹⁴, —NR¹⁴C(O)R¹⁴,—NR¹⁴C(S)R¹⁴, —NR¹⁴C(O)OR¹⁴, —NR¹⁴C(S)OR¹⁴, —NR¹⁴C(O)NR¹⁵R¹⁵,—NR¹⁴C(NR¹⁴)R¹⁴ and —NR¹⁴C(NR¹⁴)NR¹⁵R¹⁵, where R¹³, R¹⁴ and R¹⁵ are aspreviously defined.

Substituent groups from the above lists useful for substituting otherspecified groups or atoms will be apparent to those of skill in the art.

The substituents used to substitute a specified group can be furthersubstituted, typically with one or more of the same or different groupsselected from the various groups specified above.

“Protecting group” refers to a group of atoms that, when attached to areactive functional group in a molecule, masks, reduces or prevents thereactivity of the functional group. Typically, a protecting group may beselectively removed as desired during the course of a synthesis.Examples of protecting groups can be found in Greene et al., ProtectiveGroups in Organic Chemistry, 3rd Ed., 1999, John Wiley & Sons, NY andHarrison et al., Compendium of Synthetic Organic Methods, Vols. 1-8,1971-1996, John Wiley & Sons, NY. Representative amino protecting groupsinclude, but are not limited to, formyl, acetyl, trifluoroacetyl,benzyl, benzyloxycarbonyl (“CBZ”), tert-butoxycarbonyl (“Boc”),trimethylsilyl (“TMS”), 2-trimethylsilyl-ethanesulfonyl (“TES”), trityland substituted trityl groups, allyloxycarbonyl,9-fluorenylmethyloxycarbonyl (“FMOC”), nitro-veratryloxycarbonyl(“NVOC”) and the like. Representative hydroxyl protecting groupsinclude, but are not limited to, those where the hydroxyl group iseither acylated or alkylated such as benzyl and trityl ethers, as wellas alkyl ethers, tetrahydropyranyl ethers, trialkylsilyl ethers (e.g.,TMS or TIPPS groups) and allyl ethers.

“Prodrug” refers to a derivative of an active phorbol compound (drug)that requires a transformation under the conditions of use, such aswithin the body, to release the active phorbol compound. Prodrugs arefrequently, but not necessarily, pharmacologically inactive untilconverted into the active drug. Prodrugs are typically obtained bymasking a functional group in the phorbol compound believed to be inpart required for activity with a progroup (defined below) to form apromoiety which undergoes a transformation, such as cleavage, under thespecified conditions of use to release the functional group, and hencethe active phorbol compound. The cleavage of the promoiety may proceedspontaneously, such as by way of a hydrolysis reaction, or it may becatalyzed or induced by another agent, such as by an enzyme, by light,by acid or base, or by a change of or exposure to a physical orenvironmental parameter, such as a change of temperature. The agent maybe endogenous to the conditions of use, such as an enzyme present in thecells to which the prodrug is administered or the acidic conditions ofthe stomach, or it may be supplied exogenously.

“Progroup” refers to a type of protecting group that, when used to maska functional group within an active phorbol compound to form apromoiety, converts the drug into a prodrug. Progroups are typicallyattached to the functional group of the drug via bonds that arecleavable under specified conditions of use. Thus, a progroup is thatportion of a promoiety that cleaves to release the functional groupunder the specified conditions of use.

“Phorbol” refers to diterpenoid compounds based on the polycyclicstructures of formulas (II) and (III):

wherein one or more substituents can be present on each of rings A, B, Cand D. Substituents can be any substituents typically present on phorbolcompounds, including but not limited to, hydroxyl, alkyl, heteroalkyl,alkoxy, hydroxyalkyl, arylalkoxy, alkoxycarbonyl, aldehyde, acyloxy, andcombinations of these groups. The numbering of atoms in the corepolycyclic structure of the phorbol compounds will follow thoseindicated above. Exemplary phorbols include compounds based on thefollowing structures:

“Phorbol ester” refers to phorbol compounds substituted with any acyloxygroup, with exemplary phorbol esters having esters at the 12 and/or 13positions of the compounds based on structural formula (II) and at the3, 4 and 5 positions of the compounds based on structural formula (III).

“Phorbol receptor” refers to a biological moiety that binds phorbolcompounds. The biological moiety may bind to the phorbol compound invitro, where the biological moiety is in a crude, semi-purified, orpurified form. In various other embodiments, the biological moiety ispresent in vivo. The binding can occur independently or in cooperationwith other components that associate with the biological moiety.Generally, the binding of the phorbol compound will have sufficientspecificity to elicit a biological activity and/or interfere with thebinding of other agents that interact with the biological moiety in asimilar or identical region bound by the phorbol compounds.

“Tumor promoter” refers to an agent, such as a compound or composition,that in classical studies of carcinogenesis is able to increase thesensitivity of tumor formation by a previously applied primarycarcinogen, but which generally does not efficiently induce tumors whenused alone. However, tumor promoters may display carcinogenic propertieswhen tested under more stringent or sensitive conditions. Exemplarytumor promoters are the compounds found in croton oil, activeingredients of which are believed to be phorbol esters and variationsthereof.

“Non-tumor promoting phorbol” refers to a phorbol compound that displaysat least one of the biological activities of phorbol compounds, such asbinding to phorbol receptors, but which do not show tumor promotingproperties. Exemplary “non-tumor promoting” phorbol compounds include,but are not limited to, 12-deoxyphorbol 13-acetate (i.e., prostratin),12-dexoxyphorbol 13-propanoate, and 12-dexoxyphorbol 13-phenylacetate.

“Biological conditions” refer to any condition that is a physiologicalcondition present in vivo, or an in vitro condition that mimics thephysiological condition. For the purposes herein, the phrase“hydrolysable under biological conditions” refers to a physiologicalcondition that results in removal of the progroup moiety to produce thebiologically active compound. Thus, where removal of the progroup occursby action of an enzyme, the biological condition comprises such enzymeto cause removal of the progroup. Where removal of the progroup occursspontaneously, such as by hydrolysis, the biological condition comprisesthe physiologic condition that results in spontaneous loss of theprogroup.

5.2 Analogs and Derivatives of Phorbol Compounds

The phorbol compounds of the present disclosure are prodrugs that aremore soluble in various mediums for administration than the active formof the phorbol compounds. In the embodiments disclosed herein, thephorbol compounds are generally based on a diterpenoid compoundcomprising a “core” polycyclic structures according to structuralformulas (II) and (III):

As noted above, one or more substituents can be present on each of ringsA, B, C, and D. Typical substituents include, but are not limited to,hydroxyl, hydroxyalky, alkyl, heteroalkyl, alkoxy, arylalkoxy,alkoxycarbonyl, aldehyde, acyloxy and combinations of these groups (see,e.g., Naturally Occurring Phorbol Esters, F. J. Evans ed., 1986, CRCPress, Boca Raton, Fla.). In some embodiments, the phorbol compounds arebased on the structural formulas (IIa) and (IIIa):

Various derivatives and analogs of the above compounds occur naturallywith various substituents present on rings A, B, C, and D, and have alsobeen made by synthetic methods. The compounds can also have additionalmodifications of the substituents shown in the parent structure. Thus insome embodiments, the phorbol compounds herein relate to compounds ofthe structural formulas (IIb), (IIc), and (IIIb):

wherein R¹, R², R³, and R⁴ is, independently of one another, selectedfrom a hydrogen or an acyl —C(O)R″, where R″ is hydrogen, or substitutedor unsubstituted alkyl, aryl, arylalkyl, heteroalkyl, heteroaryl, orheteroarylalkyl as defined herein. In some embodiments, the R″ isselected from methyl, unsaturated lower alkyl, lower n-alkyl, benzyl,4-hydroxybenzyl, 4-methoxybenzyl, and 4-acetyloxybenzyl. In someembodiments, an oxygen atom is added to the double bond between carbonatoms 6 and 7 such that the phorbol compound has an epoxide in ring B.In various embodiments, the phorbol compounds in the present disclosureare active phorbol compounds. As used herein, active phorbol compoundrefers to a phorbol displaying at least one of the biological activitiesassociated with phorbol compounds, as further described herein (e.g.,binding to a phorbol receptor, tumor promotion, anti-viral activity,etc). Without being bound by theory, active phorbol compounds with an—OH substituent on position 4 carbon have been shown to be in the βconformation while inactive compounds are in the α conformation (see,e.g., Silinsky et al., 2003, Br J Pharmacol. 138(7):1191-201).

In some embodiments, exemplary compounds of the present disclosure areanalogs and derivatives of the following phorbol compound:

The phorbol compounds disclosed herein also include analogs andderivatives of 12-deoxyphorbol based on the following structure:

In other embodiments, the phorbol compounds disclosed herein are analogsand derivatives of ingenol compound based on the following structure:

As note above, additional substituents can be present on rings A, B, C,and D, along with modifications to the hydroxyl and/or methyl groupspresent throughout the phorbol structure. The numbering of thesubstituents follows the convention given above. In some embodiments,the compounds herein comprise esters of the phorbol compounds, where theester is present on any of the rings or the substituents of the parentphorbol structures. Many phorbol compounds based on structural formula(II) have ester groups on the 12-carbon and/or 13-carbon position of thecompounds above. Exemplary phorbol esters include, but are not limitedto, 12-deoxyphorbol 13-acetate, 12-deoxyphorbol 13-phenylacetate;12-deoxyphorbol 13-propanoate; 12-deoxyphorbol 13-isobutyrate;12-deoxyphorbol 13-myristate; 12-deoxyphorbol 13-tetradecanoate;12-deoxyphorbol 13-angelate; phorbol 13-acetate; phorbol 13-myristate;phorbol-12,13-diacetate; phorbol-12,13-dibutyrate;phorbol-12,13-didecanoate; phorbol-12-myristate 13-acetate;phorbol-12,13-dibenzoate; and phorbol-12-acetate 13-tigliate. Phorbolcompounds based on structural formula (III) have esters and the 3, 4 and5 positions, and in some embodiments, the 20 carbon position. Exemplaryphorbol esters of the ingenol compounds for the prodrugs herein include,but are not limited to, ingenol 3 acetate, ingenol-3-propionate, ingenol3-palmitate, ingenol-3-angelate, and ingenol 3-benzoate. Other phorbolesters that are suitable for the purposes herein will be apparent to theskilled artisan.

In some aspects, the phorbol compounds are prodrug compounds accordingto structural formula (I):R—X—O—C(O)—R′  (I)

including salts and hydrates thereof, wherein:

R is a residue of a phorbol ester;

X is an alkylene chain containing from 1 to 12 carbon atoms;

R′ is a moiety that either bears a permanent charge or that is ionizableat a pH in the range of about to about 2.0-8.0, a pH in the range ofabout 4.0-7.4, or a pH in the range of about 6.8-7.4;

the illustrated —X—O—C(O)—R′ group is linked to the 6-carbon of R; and

the illustrated —X—O—C(O)—R′ group hydrolyzes under biologicalconditions to yield a group of the formula —X—OH.

In some embodiments, the X of structural formula I is a methano (—CH₂—).The R′ is a group that comprises a carboxyl group, such as acarboxyl-substituted lower alkyl, or a salt thereof.

In some embodiments, the R′ is a group of the formula —(CH₂)_(m)—C(O)OM,where M is hydrogen or a counter ion and m is an integer ranging from 1to 4. The counter ion can be any ion that can form an ionic bond withthe ionized oxygen atom of the carboxyl group. Exemplary counter ionsinclude, but are not limited to, Na⁺ or K⁺ or an organic base such asethanolamine, diethanolamine, triethanolamine, morpholines, as furtherdescribed below.

In some embodiments, the R′ of the compounds further comprises an aminogroup. In some embodiments, the amino group can be an amino acid. Insome embodiments, R′ can be selected from—(CH₂)_(n)—CH[(CH₂)_(n)—NH₂]—(CH₂)_(n)—C(O)OM and—(CH₂)_(n)—CH(NH₂)—(CH₂)_(n)—C(O)OM, where M is as defined as above andeach n is, independently of the others, an integer ranging from 0 to 4.

In other embodiments, the R′ of structure (I) is a group of the formula—Y—Z, wherein:

Y is a branched or unbranched, saturated or unsaturated alkylene chaincontaining from 1 to 4 carbon atoms;

Z is selected from —C(O)OM, —NR^(b)R^(b) and —NR^(c)R^(c)R^(c);

M is hydrogen or a counter ion;

each R^(b) is, independently of the other, selected from hydrogen, loweralkyl, lower hydroxyalkyl and lower alkoxyalkyl, heteroalkyl or,alternatively, two R^(b) groups bonded to the same nitrogen atom can betaken together with the nitrogen atom to which they are bonded to form a5- to 7-membered heteroatomic ring (e.g., cycloheteroalkyl),

each R^(c) is, independently of the others, selected from lower alkyl,lower hydroxyalkyl and lower alkoxyalkyl.

In some embodiments of the phorbol compounds above,

any of the alkyl group or moiety is a branched or unbranched alkanyl;

Y is an alkano;

each R^(b) is, independently of the other, selected from hydrogen andlower alkanyl;

each R^(c) is, independently of the others, selected from lower alkanyl;and

NR^(b)R^(b) is selected from morpholinyl, N-morpholinyl, piperazinyl,1-piperazinyl, 1-methyl-piperazinyl and 1-methyl-4-piperazinyl. The Ycan be selected from methano, ethano, propano and butano.

In some embodiments, independently the Z is —C(O)OM or selected fromdimethyl amino, diethylamino, and N-morpholinyl.

In various embodiments described above, the core structure R can beselected from (R1) and (R2):

wherein:

R″ is selected from (C₁-C₁₄) alkyl and benzyl.

In some embodiments of compounds (R1) and (R2), the R″ is selected frommethyl, unsaturated lower alkyl, lower n-alkyl, benzyl, 4-hydroxybenzyl,4-methoxybenzyl, and 4-acetyloxybenzyl.

In some embodiments, the core structure R can be selected from (R3):

wherein:

R″ is selected from an alkyl, unsaturated lower alkyl, and benzyl.

In some embodiments of compounds (R3), the R″ is selected from methyl,branched alkenyl, lower n-alkyl, benzyl, 4-hydroxybenzyl,4-methoxybenzyl, and 4-acetyloxybenzyl.

In further embodiments, the compounds have the structures (Ia) or (Ib):

including the salts and hydrates thereof, wherein:

R″ is selected from lower substituted or unsubstituted alkyl,substituted or unsubstituted alkenyl, and benzyl.

In some embodiments of the compounds of structure (Ia) and (Ib), R′ canbe a group that comprises a carboxyl group, such as acarboxyl-substituted lower alkyl, or a salt thereof. The carboxyl groupcan be of the formula —(CH₂)_(m)—C(O)OM, where M is hydrogen or acounter ion and m is an integer ranging from 1 to 4.

In other embodiments of compound (Ia) and (Ib), the R′ can comprise anamino group, such as an amino acid. Thus, the R′ can be selected from—(CH₂)_(n)—CH[(CH₂)_(n)—NH₂]—(CH₂)_(n)—C(O)OM and—(CH₂)_(n)—CH(NH₂)—(CH₂)_(n)—C(O)OM, where M is as defined as above andeach n is, independently of the others, an integer ranging from 0 to 4.

In some embodiments of compounds (Ia) and (Ib), the R′ is a group of theformula —Y—Z, wherein:

Y is a branched or unbranched, saturated or unsaturated alkylene chaincontaining from 1 to 4 carbon atoms;

Z is selected from —C(O)OM, —NR^(b)R^(b) and —NR^(c)R^(c)R^(c);

M is as defined as above;

each R^(b) is, independently of the other, selected from hydrogen, loweralkyl, lower hydroxyalkyl, lower alkoxyalkyl, and heteroalkyl or,alternatively, two R^(b) groups bonded to the same nitrogen atom can betaken together with the nitrogen atom to which they are bonded to form a5- to 7-membered heteroatomic ring; and

each R^(c) is, independently of the others, selected from lower alkyl,lower hydroxyalkyl and lower alkoxyalkyl.

As described previously, in the embodiments described above, thecompound can have the features selected from:

any alkyl group or moiety is a branched or unbranched alkanyl;

Y is an alkano;

each R^(b) is, independently of the other, selected from hydrogen andlower alkanyl;

each R^(c) is, independently of the others, selected from lower alkanyl;and

NR^(b)R^(b) is selected from morpholinyl, N-morpholinyl, piperazinyl,1-piperazinyl, 1-methyl-piperazinyl and 1-methyl-4-piperazinyl.

In some embodiments, Y is selected from methano, ethano, propano andbutano.

Thus, in some embodiments, Z is selected from —C(O)OM, dimethylamino,diethylamino, trimethylamino, triethylamino, and N-morpholinyl.

In some embodiments of the structures R1, R2, R3, (Ia) and (Ib), R″ canbe selected from methyl, ethyl, propyl, butyl, pentyl, and benzyl.

In some embodiments where —NR^(b)R^(b) is in protonated form or—NR^(c)R^(c)R^(c), the compound can further comprise a anioniccounterion. Exemplary anionic counterions include, but is not limitedto, Cl⁻, Br⁻, carboxylate ion, sulfonate ion, sulfate ion, acetate,oxalate, maleate, fumarate, methanesulfonate, or toluenesulfonate. Othersuitable counterions will be apparent to the skilled artisan.

In the present disclosure, the compounds can be identified by eithertheir chemical structure or their chemical name. When the chemicalstructure and the chemical name conflict, the chemical structure isdeterminative of the identity of the specific compound. Furthermore, thecompounds described herein, as well as the various compound speciesspecifically described and/or, illustrated herein, may exhibit thephenomena of tautomerism, conformational isomerism, geometric isomerismand/or optical isomerism. For example, the compounds and prodrugs caninclude one or more chiral centers and/or double bonds and as aconsequence can exist as stereoisomers, such as double-bond isomers(i.e., geometric isomers), enantiomers and diasteromers and mixturesthereof, such as racemic mixtures. As another example, the compounds andprodrugs can exist in several tautomeric forms, including the enol form,the keto form and mixtures thereof. As the various compound names,formulae and compound drawings within the specification and claims canrepresent only one of the possible tautomeric, conformational isomeric,optical isomeric or geometric isomeric forms, it should be understoodthat the compound encompasses any tautomeric, conformational isomeric,optical isomeric and/or geometric isomeric forms of the compounds orprodrugs having one or more of the utilities described herein, as wellas mixtures of these various different isomeric forms.

In various embodiments, depending upon the nature of the varioussubstituents, the phorbol compounds of the present disclosure can be inthe form of salts. Such salts include salts suitable for pharmaceuticaluses (“pharmaceutically-acceptable salts”), salts suitable forveterinary uses, etc. Such salts can be derived from acids or bases, asis well-known in the art. Generally, pharmaceutically acceptable saltsare those salts that retain substantially one or more of the desiredpharmacological activities of the parent compound and which are suitablefor administration to humans. Pharmaceutically acceptable salts includeacid addition salts formed with inorganic acids or organic acids.Inorganic acids suitable for forming pharmaceutically acceptable acidaddition salts include, by way of example and not limitation,hydrohalide acids (e.g., hydrochloric acid, hydrobromic acid, hydriodic,etc.), sulfuric acid, nitric acid, phosphoric acid, and the like.Organic acids suitable for forming pharmaceutically acceptable acidaddition salts include, by way of example and not limitation, aceticacid, trifluoroacetic acid, propionic acid, hexanoic acid,cyclopentanepropionic acid, glycolic acid, oxalic acid, pyruvic acid,lactic acid, malonic acid, succinic acid, malic acid, maleic acid,fumaric acid, tartaric acid, citric acid, palmitic acid, benzoic acid,3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid,alkylsulfonic acids (e.g., methanesulfonic acid, ethanesulfonic acid,1,2-ethane-disulfonic acid, 2-hydroxyethanesulfonic acid, etc.),arylsulfonic acids (e.g., benzenesulfonic acid, 4-chlorobenzenesulfonicacid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid,camphorsulfonic acid, etc.),4-methylbicyclo[2.2.2]-oct-2-ene-1-carboxylic acid, glucoheptonic acid,3-phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid,lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoicacid, salicylic acid, stearic acid, and muconic acid.

Pharmaceutically acceptable salts also include salts formed when anacidic proton present in the parent compound is either replaced by ametal ion (e.g., an alkali metal ion, an alkaline earth metal ion or analuminum ion), or coordinates with an organic base (e.g., ethanolamine,diethanolamine, triethanolamine, N-methylglucamine, morpholine,piperidine, dimethylamine, diethylamine, etc., and the like).

5.3 Synthesis of Analogs and Derivatives

The compounds disclosed herein can be synthesized by a variety ofdifferent synthetic routes using commercially available startingmaterials and/or starting materials prepared by conventional syntheticmethods.

In some embodiments, phorbol and related compounds can be isolated fromvarious natural sources. Phorbol and Ingenol esters are present invarious plants of the genus Euphoriaceae, including the Spurge andPoinsettia families (see, e.g., Naturally Occurring Phorbol Esters, F.J. Evans ed., 1986, CRC Press, Boca Raton, Fla.). Exemplary Euphorbiaplants from which phorbol compounds have been isolated include, amongothers, Euphorbia Fischeriana (Liu et al., 1996, Chinese ChemicalLetters 7(10):917-918), Homalanthus nutans, and Homolanthus acuminatus;Neoboutonia melleri (Zhao et al., 1988, Phytochemistry 48(7):1173-1177);Excoecarcia agallocha (Erickson et al., 1995, J. Natural Products58(5):769-72); and Croton californicus (Chavez et al., 1982, J. NaturalProducts 45(6):745-8). Phorbol epoxides of the structure R2 can also beobtained from natural sources. For example, Zayed et al., Experientia(1977), 33(12), 1554-5 describes the isolation of R2 from the Pimeleaprostrata and Pimelea simplex plants. Various phorbol compounds,including 12-deoxyphorbol 13-acetate, are also available commercially(e.g., LC Laboratories, Woburn, Mass., USA; and Alexis, Switzerland) andcan be used for the synthesis of the compounds disclosed herein.Exemplary plants from which ingenol esters can be obtained include, butare not limited to, Euphorbia antiquorum, Euphorbia peplus, Euphorbiaingens and Euphorbia peplus (see, e.g., Rizk et al., 1985,Phytochemistry 24:1605-6).

Methods for isolating phorbol compounds from natural sources will beapparent to the skilled artisan. For instance, an exemplary method forisolating 12-dexoxyphorbol 13-acetate (i.e., prostratin) is described inU.S. Pat. No. 5,599,839, incorporated herein by reference. Generally,plant extracts can be subjected to an alcohol and alcohol/organicsolvent extraction and then processed by various partition methods(e.g., solvent partition). Additional purification can be obtained withvarious other techniques, including, among others, crystallization andchromatographic separation, such as gel exclusion, ion exchange, andHPLC chromatography.

Synthetic methods for producing phorbol parent compounds and relatedderivatives are also described in the art. Complete synthesis of phorbolcan use Diels-Alder cycloaddition combined with aldol condensation(Wender et al., 1987, J. Am. Chem. Soc. 109:4390). Phorbol synthesisusing oxidopyrilium [5+2] cycloaddition and zirconium mediatedenyne-cyclization has also been described (Wender et al., 1989, J. Am.Chem. Soc. 111:8954; Wender et al., 1990, J. Am. Chem. Soc. 112:4959;Wender et al., 1997, J. Am. Chem. Soc. 119:12976). Assymetric synthesisof phorbol is given in Wender et al., 1997, J. Am. Chem. Soc. 119:7897.Another synthetic route using an intramolecular nitrile oxide [3+2]cycloaddition combined with an aldol condensation is described in Sugitaet al., 1995, Tetrahedron Lett. 36:1067. Other references describingsynthetic routes for phorbol compounds include Paquette et al., 1984, J.Am. Chem. Soc. 106:1446-1454; Rigby et al. J. Org. Chem. 55:2959-2962;Carroll et al., 2000, Org. Lett. 2(18):2873-2876; and Cha et al., 2001,J Am Chem Soc 123, 5590. Methods for synthesizing various derivatives ofphorbol compounds are disclosed in, among others, U.S. Pat. Nos.6,080,784, 5,962,498, 5,955,501, 5,643,948, 5,145,842, WO 96/40614.Complete synthesis of ingenols are described in Nickel et al., 2004, JAm Chem. Soc. 126:16300 and Winkler et al., 2002, J Am Chem. Soc. 124(33), 9726-9728. All publications incorporated herein by reference.

A variety of exemplary synthetic routes that can be used to synthesizethe prodrug forms of the phorbol compounds are illustrated in Scheme(I).

The synthetic methods of Scheme (I) generate the prodrug forms of12-deoxyphorbol 13-acetate, either as the carboxylic acid or the aminoacid. Generally, a dicarboxylic acid HOOC(CH₂)_(m)COOH is reacted in thepresence of a coupling agent, such as an acylation catalyst4-dimethylaminopyridine (DMAP) (see, e.g., U.S. Pat. No. 5,663,335,incorporated herein by reference). The coupling agent activates thedicarboxylic acid to promote reaction with the hydroxyl group. Use of acyclic anhydride, substituted or unsubstitued, such as butanedioicanhydride (i.e., succinic anhydride) for the carboxylation also allowsfacile synthesis of the carboxylated phorbol. Where cyclic anhydridesare used, the coupling agent typically activates one of the two carbonylcarbons, promoting its attack by the hydroxyl group of the phorbolcompound. Non-limiting examples of various cyclic anhydrides include,among others, substituted or unsubstituted 2-butendioic anhydride,pentanedioic anhydride, and hexanedioic anhydride.

Analogously, amino substituted R′ of structural formula (I) can besynthesized using substituted dicarboxylic acidsHOOC—(CH₂)_(n)—CH[(CH₂)_(n)—NH₂]—(CH₂)_(n)—COOH orHOOC—(CH₂)_(n)—CH(NH₂)—(CH₂)_(n)—COOH for generating the compounds inwhich the R′ is —(CH₂)_(n)—CH[(CH₂)_(n)—NH₂]—(CH₂)_(n)—COOH or—(CH₂)_(n)—CH(NH₂)_(n)—(CH₂)_(n)—COOH. Use of substituted cyclicanhydride, as shown in Scheme (1), provides another method ofsynthesizing the amino substituted derivatives of the disclosed phorbolcompounds.

Various amino ester derivatives can be prepared by reacting the phorbolstarting compound with an amine substituted carboxylic acid in presenceof a coupling reagent (e.g., DCC) and an acylating catalyst (e.g., DMAP)to synthesize the compound of structural formula (I) in which the R′ is—Y—Z, wherein the Y is a branched or unbranched, saturated orunsaturated alkylene chain, and Z is the substituted amine. Use of adisubstituted amine yields the tertiary amine —NR^(b)R^(b) while atrisubstituted amine yields the permanently charged quartenary amine—NR^(c)R^(c)R^(c). Non-limiting examples of amine substituted carboxylicacids useful in synthesis of the aminoester derivatives include, amongothers, N′N′-dimethylglycine, N′N′-dimethylproprionate,N′N′-dimethylbutanoate, N′N′N′-trimethylglycine,N′N′N′-trimethylpropionate, and N′N′N′-trimethylbutanoate. Other aminesubstituted carboxylic acids will be apparent to the skilled artisan.

For the disclosed phorbol compounds of structure (I) in which R′ is—Y—Z, where Z is NR^(b)R^(b) and the two R^(b) groups are bonded to thesame nitrogen to form a 5-7 heterocyclic ring, suitable carboxylic acidsof morpholine, piperizine, pyrrolidine, and piperidine can be used.Non-limiting examples of heterocyclic carboxylic acids include, amongothers, 4-pyrrolidinobutyric acid and 5-morpholinopentanoic acid. Otherheterocylic carboxylic acids will be apparent to the skilled artisan.

Where reactive groups on the phorbol compound require protection, theycan be protected with a suitable protecting group. Oxygen atoms ofhydroxyl groups can be protected in the form of acyl, benzyl,trialkylsilane, benzyloxycarbonyl, 4′-methoxyphenyldiphenylmethyl andtrimethylsilylyethoxycarbonyl groups (Greene et al., Protective Groupsin Organic Chemistry, 3rd Ed., 1999, John Wiley & Sons, NY). Thesegroups have varying stabilities to or removed under acidic, basic orreducing conditions or with fluoride ion reagents, depending on the typeof protecting group. Carbonyl functions can be protected by conversionto acetals or ketals, or by reduction to the alcohol level followed byprotection with standard protecting groups for the hydroxy group. Thehydroxy group at 2-position carbon of the phorbol compounds can becapped by reaction with substituted or unsubstituted alkyl, aryl, oraralkyl isocyanate in the presence of a catalyst such as dibutyltindilaurate. For example, the oxygen atoms at position 3, 4, 12, and 13 ofthe parent phorbol compound or positions 3, 4, and 13 of the parent12-deoxyphorbol compound can be blocked with a suitable protecting groupchosen from the oxygen atom protecting groups described above. Othermethods of protecting functional groups for synthesizing the compoundsherein will be apparent to the skilled artisan.

5.4 Uses of the Compounds

Phorbol compounds display a myriad of biological activities, including,but not limited to, tumor promotion, modulation of signal transductionpathways, activation of latent viruses, inhibition of virus entry,modulation of inflammatory responses, modulation of cell proliferation,and effects on nociception. The phorbol compounds described herein areuseful in modulating any of these activities associated with phorbolcompounds. Moreover, the phorbol compounds find uses in the treatment ofvarious disorders and diseases associated with the biological activitiesmodulated by phorbol compounds. Thus, the prodrug compounds can findapplications in treating inflammatory reactions, neoplastic growth,viral infections (e.g., HIV infection), and for use as analgesics (i.e.,inhibiting nociception). For embodiments involving treatment of asubject, phorbol compounds that are either non-tumor promoting or haveminimal tumor promoting activity are desirable. In some embodiments, thenon-tumor promoting compound can be derivatives and analogs that inhibitthe tumor promoting activity of tumor promoting phorbol compounds. Inthis regard, phorbol compounds in which the R″ of the structuresdisclosed herein (e.g., structure 1a; RI; R2) are lower alkanyl, lowerunsubstituted alkyl, or benzyl have been shown to display minimal or notumor promoting activity for the active compounds.

Generally, the phorbol compounds disclosed herein are prodrugs in thatthe compounds are converted to the active forms. This can occur throughinherent instability of the prodrug moiety under a specified set ofconditions or through the action of a biological process thatbiotransforms the prodrug to the active drug metabolite. Thus, for manyof the uses herein, the phorbol compound is subjected to biologicalconditions that result in conversion of the prodrug compounds to theactive form by removal of the progroup moiety.

The compounds can be used independently as a single agent, or in someembodiments, used in combination with other agents. These combinationsinclude other phorbol compounds as well as other non-phorbol agents.Thus, in various embodiments, the phorbol compounds can be usedadjunctively with other anti-inflammatory, anti-retroviral,anti-nociceptive, and anti-neoplastic compounds.

5.4.1 Binding and Modulation of Phorbol Receptors and Associated SignalTransduction Pathways

In accordance with the above, in some aspects, the phorbol compounds canbe used to modulate the activity of phorbol receptors and theircorresponding signal transduction systems by contacting a phorbolreceptor with the compounds disclosed herein and measuring the activityof the phorbol receptor. A phorbol receptor is any biological moietythat binds to phorbol compounds with specificity and/or affects aphysiological process associated with the receptor when the bindingoccurs in vivo or in vitro. Compounds that bind a phorbol receptorinclude agonists and antagonists of other phorbol compounds or similarmodulators, for example diacyglycerol. Antagonists include compoundsthat bind the phorbol receptor but which itself is without any directeffect on a cellular process affected by the phorbol receptor. Bindinginteractions can take place in crude extracts containing the receptor,or semi-purified or purified receptor preparations. Binding is readilymeasured with labeled phorbol compounds, such as radiolabeled phorbolcompounds, which can be made during synthesis by incorporating labeledstarting materials or intermediates, or by exchange reactions. Suitableradiolabels include ³H, ¹⁴C, ³²P, ³⁵S, ³⁶Cl, ⁵⁷Co, ¹³¹I and ¹⁸⁶Re. Otherlabeling methods will be apparent to the skilled artisan.

In some embodiments, the phorbol compounds can be used to modulate theactivity of receptors protein kinase C, a family of lipid regulatedserine threonine kinases that phosphorylate numerous cellular proteins.The PKC family comprises at least three subclasses of structurallyrelated proteins, which are categorized based on their regulation. Theclassical PKCs (cPKCs) encompass isoforms whose activity is regulated byCa⁺² and/or lipid diacylglycerol, and modulated by phorbol esters.Exemplary cPKCs include PKCα, β1, βII, and γ. A second class of PKCs,designated in the art as novel PKCs or nPKCs, are also activated bydiacylglycerol and phorbol esters but does not require Ca⁺². ExemplarynPKCs include δ, ε, θ and η forms. The third class of PKCs, denoted inthe art as atypical PKCs are not regulated by Ca⁺² and do not respond toeither diacylglyercol or phorbol esters. Exemplary atypical PKCs includePKCζ and PKCτ/λ. Because the third class of PKCs appears not to interactwith phorbol esters, these proteins would not be characterized asphorbol receptors.

Another group of phorbol receptors are defined by a class of proteinsdescribed as PKC related kinases. These groups of receptors are serinethreonine kinases regulated by diacylglyercol and phorbol esters.Exemplary receptors of this class include PKCμ, PKD and PKRs. Thesekinases differ from the other PKCs with respect to substrate specificityand regulation. Forms PKCμ/PKD contain a putative transmembrane domainin the amino terminal region and a C1 region with two cyteine richdomains that bind phorbol esters, and a pleckstrin homology domain. Thekinase catalytic domain is related to the kinase of Ca⁺²/calmodulindependent kinase II. This enzyme does not act on substrates typicallyactive with other PKCs but acts on substrates phosphorylated bycalmodulin dependent kinases. The second type of PKC-related kinases,referred to as PRKs, has a kinase region with homology to the kinasedomain of PKCs, but appears to not bind phorbol esters or regulated byCa⁺. Instead, kinase activity is sensitive to phospholipidsphosphatidylinositol 4,5 bisphosphate and phosphatidylinositol 3,4,5,triphosphate.

In other embodiments, the phorbol receptors are class of proteinsdescribed as “nonkinase phorbol ester/DAG receptors.” These receptorsinclude the mammalian α and β chamaerins, Ras-GRP, and Caenorhabditiselegans Unc-13. Nonkinase phorbol receptors are characterized by asingle copy of the cysteine rich domain that appears to function inbinding to diacylglycerol. The chimaerin proteins have a regulatorydomain found in PKCs and BCR, and displays phorbol binding propertysimilar to PKCα. Chimaerin do not have sequences associated with kinaseactivity, but have a GAP (GTPase activating protein) at the carboxyterminal region, which may function in down regulating Rac function.Another member of the non-kinase phorbol esters is RasGRP (i.e., Rasguanyl-releasing protein). RasGRP has a single cysteine rich region atthe carboxy terminal domain similar to those in other PKCs, a catalyticregion with sequence similarity to CDC25 box, which acts as a Rasactivator, and a Ras exchange motif conserved among guanyl nucleotidereleasing factors. In the RasGRP, the phorbol ester binding site mayfunction in recruitment of RasGRP to the plasma membrane. Binding tothese phorbol receptors and subsequent activation of PKC may be used toassess the activity the phorbol compounds described herein.

It is to be understood that the descriptions of phorbol receptors abovereflect the state of knowledge in the art and are not intended to belimiting. Thus while some biological moieties may be currently describedas not binding phorbol compounds, there may be subsequent evidenceshowing that such biological moieties bind to phorbol compounds, andthus would be encompassed within the description of a phorbol receptor.

Many of the PKC target substrates are components of signal transductionpathways and include proteins that regulate ion channels, calcium- andcalmodulin-binding proteins, growth factor receptors, structural andregulatory proteins of the cytoskeleton, components of thetranscriptional machinery, efflux pumps, and many other proteins. PKCphosphorylates serine and threonine residues on these protein targets,typically in a consensus sequence RxxS/TxRx (where x is any amino acid)(Nishikawa et al., 1997, J. Biol. Chem. 272:952-960). Any of the knownsubstrates, natural or synthetic, can be used to detected phorbolmediated activation of PKC activity. Exemplary substrate targets includeMARCKS, GAP43, GABA type A receptor g2L, EGF receptor, ribosomal proteinS6, Troponin I, insulin receptor tyrosine kinase, c-Kit (stem cellfactor receptor), annexin II, and Wiskott-Aldrich interacting protein.Exemplary synthetic substrates are described in Nishikawa et al., supra;Loog et al., 2005, J. Biomol. Screen. 10(4):320-328; and Toomik et al.,1997, Biochem. J. 322:455-460. The prodrug forms can be tested in thepresence of agents capable of converting the prodrug forms to the activecompounds. Exemplary agents for activation include, culture medium(conditioned or unconditioned), cell/issue homogenates, microsomalextracts, or cytosol preparations. The biological activating agents canbe prepared from different organs, such as the liver or intestine, whichcan contain biological activities capable of biotransforming the prodrugforms.

In other embodiments, the phorbol compounds can be used to modulate, orassessed for their ability to modulate, signal transduction pathways andother cellular process dependent on phorbol receptors (e.g., PKC). By“signal transduction or cellular process dependent on phorbol receptors”is meant any signal transduction process that is modulated, whole or inpart, by binding of phorbol compounds to receptors such as the PKCisoforms. Modulation refers to activation or inhibition of the signaltransduction process, and can be detected by examining the cellularproducts or cellular states affected by phorbol receptor activation.Processes regulated by phorbol receptor activity include, by way ofexample and not limitation, cell adhesion, ion channel activity (e.g.,calcium, sodium, and potassium channels), neurotransmitter transporters,transcriptional regulation, hormone activity, and synaptic plasticity.Thus, in some embodiments, the phorbol compounds can be used to modulateor assess transcription dependent on phorbol receptors. Promoterscontaining TPA responsive element (TRE) and serum responsive element(SRE) are known to be regulated by PKC activation. For instance,activation of PKCγ leads to dephosphorylation of c-Jun, a transcriptionregulator, which binds to TRE sequences to activate expression of theTRE regulated gene product. PKC appears to mediate this effect bystimulating dephosphorylation of glycogen synthase kinase-3β (GSK-3β),ultimately leading to dephosphorylation of c-Jun and thereby affectingits interaction with c-fos to modulate promoter activity. Gene productsmodulated via TRE containing genes include, as non-limiting examples,collagenase, stromelysin, and metallothionein IIA.

Transcriptional modulation of SRE containing promoters appears to occurthrough involvement of PKC in the MAP kinase pathway involving ERK(extracellular receptor kinase), which is activated by a number ofgrowth factor receptors. Binding of growth factors to their cognatereceptors results in activation of PKC, which in turn phosphorylatesRaf, a modulator or ERK activity. Raf is also activated by Ras, which isalso activated by growth factors. Thus, PKC appears to supplement theactivation of Raf that occurs via Ras activity. Activated ERK modulatesthe activity of SRE containing promoters by modifying TCF, atranscription factor that interacts with SRF (serum response factor) tobind to SRE sites. One consequence of increased SRE promoter activity istranscription of c-fos, a gene product involved in tumorigenesis. Thus,the JNK and RAF signaling pathways are modulated by phorbol esters, andthus comprise phorbol receptor dependent pathways.

An effect of the binding of phorbol esters to PKC is the translocationof the PKC protein from the cytosol to the membrane. Binding of naturalligand diacylglycerol or phorbol compounds to the C1 region of certainPKCs in the presence of Ca⁺² appears to make the C1 region morehydrophobic, and coupled with other conformational changes in themembrane, increases its affinity for the cell membrane. The result istranslocation of the PKC from the cytosol to the membrane, a transitionreadily detected using labeled PKC proteins (see, e.g., Sakai et al.,1997, J Cell Biol. 139(6):1465-76). In these assays, binding of activephorbol esters (i.e., phorbol compounds that stimulate PKC activity)induces translocation while inactive phorbol esters (i.e., phorbolcompounds that do not stimulate PKC activity) do not inducetranslocation. Translocation with various lipid ligands and phorbolesters are described for various PKC subtypes, such as PKCγ, PKCα, PKCξand PKCδ (see, e.g., Seki et al., 2005, Genes to Cells 10:225-239).Thus, the phorbol compounds can also be tested for effect of signaltransduction pathways by contacting cells with the phorbol compoundsunder biological conditions that result in removal of the progroup anddetermining the intracellular translocation of PKC enzymes.

It is to be understood that the pathways described above are exemplaryof signal transduction pathways dependent on phorbol receptors. Otherpathways will be apparent to the skilled artisan, and can serve asuseful targets for the phorbol compounds herein as well as providingassays for assessing phorbol activity. As noted above, phorbol activityincludes antagonistic as well as agonistic activity against variousphorbol receptors and signal transduction pathways dependent on phorbolreceptor activity.

5.4.2 Active Tumor Promoters and Tumor Promoter Inhibitors.

In various embodiments, the phorbol compounds find uses as tumorpromoters that accelerate the tumor forming ability of variouscarcinogens when administered in combination to a host. Phorbol esterswere originally identified based on their tumor promoting ability, acharacteristic thought to be related to activation of PKC signalingpathways and induction of inflammatory reactions. Classically, tumorpromoting activity assays involve topically applying the skin of a hostanimal host a tumor initiator (e.g., carcinogen such as7,12-dimethyl-benz(a)anthracene). This is followed by administration ofthe tumor promoter compound (e.g., phorbol compound), either orally ortopically. Formation of skin tumors are assessed visually as well ashistologically. Control groups, for example, treatment of tumor promoteralone or initiator alone, are used to determine the activity of thetumor promoter. Known tumor promoting phorbol compounds, such as12-O-tetradecanoylphorbol 13-acetate (TPA), serve as useful referencecompounds in assessing tumor promoting potential. Where the effect isinhibiting tumor promotion, the phorbol compound under study can be usedin combination with a known tumor promoter. For instance, in the skintest, tumorigenesis is initiated by applying the tumor initiator. Thisis followed by administration of the phorbol compound in combinationwith, simultaneously or sequentially, a known tumor promoter such asTPA.

Other in vivo assays for tumor promoting activity have been developedbased on other characteristics of tumor formation. In some embodiments,the tumor promotion assay can use the ability of tumor promoters toinduce angiogenesis in animal model systems (see, e.g., Morris et al.,1988, Am J Physiol. 254(2):C318-22). Active tumor promoters appear tostimulate angiogenesis while inactive tumor promoters do not stimulateangiogenesis. Thus, determining whether a phorbol compound promotesangiogenesis provides another basis to assess the tumor promoting ortumor promoter inhibiting activity.

In addition to in vivo systems, various in vitro based systems areavailable for assessing tumor promoting activity. In some embodiments,the in vitro assessment comprises contacting cultured cell lines thatrespond to treatment with phorbol compounds with the phorbol compoundsherein, and measuring a cellular reaction to the compound. Depending onthe cell type, cellular responses include, among others, stimulation ofproliferation of quiescent cells, inhibition of cell proliferation, andstimulation of terminal differentiation. Cultured cells that respond tophorbol compounds by proliferating include as non-limiting examples, 3T3mouse fibroblasts (Rosengurt, E., 1986, Science 234:161-166) and restingT lymphocytes (Berry et al., 1990, Eur. J. Biochem 189:205-214).Cultured cells that stop proliferating and undergo differentiation inresponse to phorbol compound include as non-limiting examples HL-60promyelocytic leukemia cell line, human T-lymphoblastic cell lineMOLT-3, and B cell Daudi cells. In these embodiments, various assays canbe used to assess cell proliferation. These include, as non-limitingexamples, incorporation of labeled DNA synthesis substrates (e.g., ³Hlabeled dNTP or digoxigenin-labeled dUTP), and labeling with DNAspecific dyes coupled with analysis in a fluorescence activated cellsorter. Other methods will be apparent to the skilled artisan.

5.4.3 Activation of Latent Viruses and Antiviral Activities: Treatmentof Viral Infections.

In some aspects, the compounds find use in attenuating or inhibitingviral activity. For purposes of treatment, a subject afflicted with aviral infection can be administered an amount of the phorbol compoundseffective to treat the viral infection. An anti-viral effect includesany effect that attenuates or inhibits viral activity and is not limitedto any mechanism of action. Thus, the anti-viral compound or compositioncan modulate, among others, viral entry into the cell, viralreplication, viral production, and virus stability. An “anti-viralcompound” refers to a compound or compositions that attenuates orinhibits viral infection.

Various phorbol compounds display anti-viral properties and display theability to activate latent viruses. “Viral latency” refers to thepersistence of a virus in non-infectious form and typically occurs wherethe virus infects the host without cytopathic effect followed by longterm maintenance of the viral genome in the host cell. Some of themechanisms for initiation of latency include, among others, infection ofnon-permissive cells, presence of conditions affecting the cytopathicprocess, or the production of viral variants. Examples of nonpermissiveinfections are Epstein Barr Virus (EBV) infection of B lymphocytes, HIVinfection of mononuclear phagocytes, cytomegalovirus (CMV) infection ofperipheral blood mononuclear cells (PMBC), and herpes simplex virus(HSV) infection of sensory neurons. Infection of the specified celltypes by the corresponding virus results in little or no expression ofviral gene products and thus no or insignificant production ofinfectious viral particles.

Maintenance of the viral genome in the latency phase depends on thevirus type. Viruses that integrate into the host genome are replicatedalong with the host chromosome, maintenance being part of host celldivision process. Retroviruses, such as HIV, propagate the viral genomeby converting the RNA into a DNA copy, which is then integrated into thehost cell genome. Other viruses are maintained episomally, where viralreplication is intimately connected to the host cell cycle butcontrolled by virally expressed proteins. EBV and papillomaviruses areexamples of viruses that exist episomally during the latency period.Still other viruses exist episomally but without replication of theviral genome. An example of this latter mechanism is persistence of HSVin sensory neurons. HSV initially infects the oral mucosa and thenenters the sensory nerve endings to establish a latent infection. Thelimited expression of certain HSV viral gene products (e.g., IE gene)limits the production of infectious viral particles. Since sensoryneurons do not replicate, there is no requirement for replication of theHSV viral genome for maintenance. There is no loss of the virus bydilution as might occur if the virus infects actively dividing cells.

Under some conditions, latent viruses undergo reactivation, a processresulting in production of viral particles from the cell harboring thelatent virus. Phorbol compounds, such as phorbol esters and12-deoxyphorbol 13-acetate, have the ability to reactivate latentviruses. Non-limiting examples of viruses showing latency include humanT cell leukemia virus type I (HTLVI) (Lin et al., 2005, Virology. June15: Epub); human immunodeficiency virus (HIV) (Korin et al., 2002, J.Virol. 76(16):8118-23); Kulkosky et al., 2001, Blood. 98(10):3006-15),and Epstein Barr Virsu (EBV) (Davies et al., 1991, J. Virol.65:6838-6844). Although, the mechanism of reactivation appears to varybetween viruses, the mechanistic aspects are not critical for theseapplications, and the compounds find a number of different uses based onreactivation activity of the phorbol compounds.

In some embodiments, the reactivation of latent viruses is used as adiagnostic test to determine the viral reservoir residing in a subjector a cell preparation. The method comprises contacting cells harboring alatent virus with an amount of phorbol compound effective to reactivatethe virus. Virus reactivation can be detected by various techniques,such as polymerase chain reaction, viral plaque assays, or virusspecific antibodies. Determining the levels of latent viruses canprovide a measure of the effectiveness of a specific viral therapy orprovide information useful in determining the effectiveness of othertherapies used to reduce the viral reservoir. In other embodiments, asfurther described below, the reactivation properties are used asadjunctive therapy in combination with other antiviral agents forreducing viral reservoirs or eliminating presence of the virus in aninfected subject. For example, prostratin appears to reactive latent HIVvirus but without affecting the effectiveness of highly activeantiretroviral therapy (HAART) (Kulkosky et al., supra). HAART is acombination drug therapy (e.g., drug cocktail) using reversetranscriptase and protease inhibitors to provide different modes ofinhibiting the virus. It is a highly effective treatment for reducingviral load and maintaining the infection in a chronic state. Treatmentscan comprise administering to a chronically infected subject a phorbolcompound displaying reactivating properties to induce the latentviruses, and adjunctively administering an anti-viral compound, such asa combination of the compounds used in HAART therapy (Kulkosky et al.,supra).

In other embodiments, the phorbol compounds can be used to treat a viralinfection by administering to a subject infected with the virus anamount of the phorbol compound effective to treat the viral infection.This use is based on the anti-viral activities of some phorbolcompounds. For instance prostratin inhibits the cytopathic activity ofHIV and also inhibits the entry of the virus into a cell (Witvrouw etal., supra). Erickson et al., 1995, J Nat. Products 58(5):769-72describes anti-viral activities of 12-deoxyphorbol13-(3E,5E-decadienoate). The mechanism of the inhibition is not criticalto the uses herein but can involve reduction in levels of the naturalcellular receptor for HIV, coreceptors CCR5 and CXCR4 (Rullas et al.,Antiviral Ther. 9:545-554). In addition to their use in the treatment ofviral infections, the compounds herein can also be used as aprophylactic measure to reduce the probability of infection by the virusin an uninfected subject.

As described throughout this disclosure, the phorbol compounds can beadministered individually or administered in combination with othertherapeutic compounds (e.g., antiviral compound), either in the form ofa composition or adjunctively by simultaneous or sequentialadministration. Thus, for treatment of retroviral infections, thephorbol compounds can be used in combination with, among others,nucleoside reverse transcriptase inhibitors, non-nucleoside reversetranscriptase inhibitors, protease inhibitors, and virusuptake/absorption inhibitors. Anti-viral compounds that affect viralfusion or viral transcription can also be used.

Nucleoside/nucleotide reverse transcriptase inhibitors inhibit action ofthe viral reverse transcriptase required for conversion of the viral RNAinto DNA during viral replication. Non-limiting examples of theseinhibitors include azidothymidine and its derivatives (e.g., AZT,Zidovudine),(2R,cis)-4-amino-1-(2-hydroxymethyl-1-1-oxathiolan-5-yl)-(1H)-pyrimidine-2-one(i.e., Lamivudine), 2′,3′-dideoxyinosine (didanosine),2′,3′-dideoxycytidine (i.e., Zalcitabine),2′,3′-didehydro-3′-deoxythymid-ine (i.e., stavudine),(1S,cis)-4-[2-amino-6-(cyclopropylamino)-9H-purin-9--yl]-2-cyclopentene-1-methanol sulfate (i.e., abacavir),(−)-beta-2′,3′-dideoxy-5-fluoro-3′-thiacytidine (i.e., emtricitabine),and phosphonate 9-R-(2-phosphonomethoxypropyl)adenine (i.e., PMPA;tenofovir disoproxil fumarate; adefovir) and various derivatives thereof(see, e.g., Deeks et al., 1998, Antimicrob. Agents Chemother.42(9):2380-2384).

Non-nucleoside reverse transcriptase inhibitors (NNRTI) are antiviralcompounds that inhibit the action of viral reverse transcriptase bybinding to the enzyme and disrupting its catalytic activity.Non-limiting examples of inhibitors of this class include11-cyclopropyl-5,11-dihydro-4-methyl-6H-dipyrido-[3,3-b-2′,3′--][1,4]diazepin-6-one (i.e., Nevirapine); piperazine,1-[3-[(1-methyl-ethyl)amino]-2-pyridinyl]-4-[[5-[(methylsulfonyl)amino]-1-H-indol-2-yl]carbonyl]-,monomethane sulfonate (i.e., Delavirdine); and(S)-6-chloro-4-(cyclopropylethynyl)-1,4-dihydro-4-(trifluoromethyl)-2H-3,-1-benzoxazine-2-one(i.e., Efavirenz). Other include quinazolinone and its derivatives, forexample trifluoromethyl-containing quinazolin-2(1H)-ones (Corbett etal., 2000, Prog. Med. Chem. 40:63-105; calanolide A (Newman et al.,1998, J Pharm. Sci. 87(9):1077-1080; and6-arylmethyl-1-(ethoxymethyl)-5-alkyluracil (i.e., emivirine) and itsanalogs (El-Brollosy, 2002, J Med. Chem. 45(26):5721-5726).

In other embodiments, the combination treatment is with proteaseinhibitors, which typically target the HIV protease enzyme, a 99-aminoacid homodimer that cleaves pol-gag polypeptides on the viral envelope.Inhibition of the HIV protease results in release of immature,noninfectious viral particles. In addition, many of the proteaseinhibitors can also exert additional antiviral effects by inhibitingcellular proteasome function. Non-limiting examples of proteaseinhibitors useful in the methods herein include indinavir, saquinavir(fortovase), ritonavir, nelfinavir, amprenavir, and lopinavir.

As noted above, HAART is a drug regimen consisting of at least threedifferent anti-retroviral drugs and is shown to be effective therapy forcontrolling viral infection and limiting the cytopathic effects of thevirus such that a chronically infected state is established in theinfected subject. In the adjunctive therapies, various combinations ofanti-retroviral agents can be used together with the phorbol compoundsdisclosed herein.

In the embodiments herein, various assays can be used to examine theanti-viral activity of the phorbol compounds. For instance, HIV viralreplication is measurable by assessing the activity of reversetranscriptase or the production of viral particles by infected cells.Reverse transcriptase measurements can be any standard assays (see,e.g., Buckheit et al., 1991, AIDS Research and Human Retroviruses7:295-302), such as an assay using labeled nucleotide triphosphates andmeasuring incorporation of label into nucleic acid. Determining thepresence of viral particles can be based on nucleic acid amplificationreactions (e.g., PCR) using viral specific primers or hybridizationreactions (e.g., microarray detection systems). Antibodies directedagainst viral specific proteins, such as capsid protein p24, are usefulin measuring the presence of viral particles. Other methods will beapparent to the skilled artisan.

Although illustrations above are for HIV, it will be apparent to theskilled artisan that analogous assays can be used for other types ofvirus. Further, it is to be understood that because the exact mechanismby which phorbol compounds exert their anti-viral activity is unclear,negative results with some assays directed to specific aspects of viralpathogenecity or negative results using certain cell types can not beindicative of the efficacy of the compounds.

In further embodiments, the phorbol compounds disclosed herein can beused in combination with non-retroviral anti-viral compounds.Combinations with non-retroviral anti-viral compounds can be applicablewhere the phorbol compound is used to reactivate latent viruses toeliminate or reduce the reservoir of virus in an infected host. The typeof non-retroviral anti-viral compound chosen will depend on the type ofvirus present in the infected host. In some embodiments, the anti-viralcompound chosen for the combination comprises an agent effective againstDNA viruses. In other embodiments, the anti-viral compound chosen forthe combination comprises an agent effective against non-retroviral RNAviruses. Exemplary anti-viral agents include, by way of example and notlimitation, acyclovir, valacyclovir, docosanol famciclovir, forcarnet,formivirsen, gangciclovir, idoxuridine, penciclovir, trifluridine,valacyclovir, vidarabine, amantadine, oseltamivir, rimantidine,zanamivir, fomivirsen, imiquimod, lamivudine, and ribavirin. Otheranti-vrial compounds for use with the phorbol compounds disclosed hereinwill be apparent to the skilled artisan.

5.5 Uses as Anti-Neoplastic and Anti-Inflammatory Agents and asAnalgesics

In some aspects, the phorbol compounds can be used to treat various cellproliferative disorders by administering to a subject afflicted with acell proliferative disorder an amount of the phorbol compound effectiveto treat the cell proliferative disorder. As used herein, a “cellproliferative disorder” refers to a condition or disease in which normalcontrols that regulate cell division are abnormal, thereby resulting inabnormal cell proliferation. Cell proliferative disorder includesneoplasms or tumors, which generally relate to abnormal growth oftissues.

Anti-neoplastic activity of certain phorbol compounds have beendemonstrated against leukemia, carcinoma, and melanoma (see, e.g., U.S.Pat. Nos. 6,063,814; 5,643,948). Cytotoxicity of 12-dexoxyphorbolcompounds against cancer cell lines are described in Fatope et al.,1996, J Med. Chem. 39(4):1005-8. Because of the effectiveness phorbolcompounds against diverse types of tumors, it has been suggested thatnon-tumor promoting compounds with anti-neoplastic activity can beeffective against many different types of cancers. Cancers aretraditionally classified based on the tissue and cell type from whichthe cancer cells originate. Carcinomas are considered as cancers arisingfrom epithelial cells while sarcomas are considered as cancers arisingfrom connective tissues or muscle. Other cancer types include leukemias,which arise from hematopoietic cells, and cancers of nervous systemcells, which arise from neural tissue. For non-invasive tumors, adenomasare considered as benign epithelial tumors with glandular organizationwhile chondromas are benign tumor arising from cartilage. In the presentdisclosure, the compounds can be used to treat proliferative disordersencompassed by carcinomas, sarcomas, leukemias, neural cell tumors, andnon-invasive tumors.

In some embodiments, the compounds can be used to treat solid tumorsarising from various tissue types, including, but not limited to,cancers of the bone, breast, respiratory tract, brain, reproductiveorgans, digestive tract, urinary tract (e.g., bladder), eye, liver,skin, head, neck, thyroid, parathyroid, and mestastatic forms thereof.Proliferative disorders of the hematopoietic system that can be treatedinclude, but are not limited to, various T cell and B cell lymphomas,non-Hodgkins lymphoma, cutaneous T cell lymphoma, Hodgkins disease, andleukemias (e.g., acute myeloid leukemia, acute lymphoblastic leukemia,chronic lymphocytic leukemia, chronic myelogenous leukemia, etc.).

As noted previously, for use of the phorbol compounds in a therapeuticsetting, prodrug forms of the compounds that are non-tumor promoting aredesirable. Non-tumor promoting compounds include, by way of example andnot limitation, 12-deoxyphorbol 13-acetate, 12-deoxyphorbol13-propanoate, and 12-deoxyphorbol 13-phenylacetate. These phorbolcompounds bind to and activate protein kinase C but do not produce thetypical tumor promoting effects (e.g., hyperplasia) of other phorbolesters (e.g., TPA), or induce only a partial response (e.g.,inflammation). Pretreatment with these agents inhibits the tumorpromoting effect of various tumor promoting phorbol esters (Szallasi etal., 1993, Cancer Res. 53(11):2507-12).

In some embodiments, the phorbol compounds can be used in combinationwith other cancer chemotherapeutic agents. For instance, U.S. Pat. No.6,063,814 describes the use of TPA in combination with cytotoxic agentaraC (cytosine arabinoside) and shows a synergistic anti-proliferativeeffect of the combination. Anti-tumor responses were also observed inpatients previously treated with hydroxyurea or bisulfan. Thus, fortreating various cell proliferative disorders, various anti-neoplasticagents can be used in combination with the phorbol compounds disclosedherein, either in the form of a composition or by adjunctiveadministration. Various classes of anti-neoplastic compounds suitablefor the uses herein include, but are not limited to, alkylating agents,antimetabolites, vinca alkyloids, taxanes, antibiotics, enzymes,cytokines, platinum coordination complexes, and substituted ureas.Exemplary alkylating agents include, by way of example and notlimitation, mechlorothamine, cyclophosphamide, ifosfamide, melphalan,chlorambucil, ethyleneimines, methylmelamines, alkyl sulfonates (e.g.,busulfan), and carmustine. Exemplary antimetabolites include, by way ofexample and not limitation, folic acid analog methotrexate; pyrmidineanalog fluorouracil and cytosine arbinoside; and purine analogsmercaptopurine, thioguanine, and azathioprine. Exemplary vinca alkyloidsinclude, by way of example and not limitation, vinblastine, vincristine,paclitaxel, and colchicine. Exemplary antibiotics include, by way ofexample and not limitation, actinomycin D, daunorubicin, and bleomycin.An exemplary enzyme effective as anti-neoplastic agent isL-asparaginase. Exemplary coordination compounds include, by way ofexample and not limitation, cisplatin and carboplatin. These and otheruseful anti-cancer compounds are described in Merck Index, 13th Ed.(O'Neil M. J. et al., ed) Merck Publishing Group (2001) and Goodman andGilmans The Pharmacological Basis of Therapeutics, 10th Edition,Hardman, J. G. and Limbird, L. E. eds., pg. 1381-1287, McGraw Hill,(1996), both of which are incorporated by reference herein.

In other aspects, the analogs and derivatives can be used to attenuateor inhibit an inflammatory reaction. Anti-inflammatory activities ofcertain phorbol compounds are described in U.S. Pat. No. 5,643,948 andMa et al., 1997, Phytochemistry 44(4), 663-666. The phorbol compoundscan be administered to a subject with an inflammatory reaction in anamount sufficient to attenuate or inhibit the inflammatory reaction. Inthese embodiments, the phorbol compounds can be use alone or incombination with other anti-inflammatory compounds, such asnon-steroidal anti-inflammatory drugs (NSAID). In some embodiments, theanti-flammatory compounds can be Cox-1 and/or Cox-2 inhibitors.Non-limiting examples of such anti-inflammatory compounds include, amongothers, aspirin, acetoaminophen, ibuprofen, diclofenac, and refocoxib.Others anti-inflammatory compounds suitable for the combinations hereinwill be apparent to the skilled artisan.

In further aspects, the phorbol compounds can be used as analgesics. Asused herein, “analgesic” refers to reduction or relief of pain.Analgesic effect of compound prostratin is described in Ma et al., 1997,Phytochemistry 44(4), 663-666. The compounds can be administered to asubject afflicted with pain an effective amount of the phorbol compoundin an amount sufficient to reduce or relieve the pain. Pain refers to anunpleasant sensory and emotional experience associated with actual orpotential tissue damage generally resulting from the stimulation ofspecialized nerve endings. Pain may be contained to a localized area, asin an injury, or it can be a more diffuse through the subject. Pain mayarise from any condition, such as physical injury, disease, or emotionaldisorder.

Various experimental tests can be used to determine the effectiveness ofthe compounds for treating pain. Art recognized tests include, amongothers, tail flick test or hot plate tests. These tests entail exposingthe tail of a test animal, typically a mouse or rat to a noxious thermalstimulus and measuring the time the animal flicks its tail in responseto the stimulus. An analgesic effect is indicated where there is anincrease in the time in which the tail flick response occurs. Othertypes of unpleasant test stimuli for measuring pain response andtolerance to pain include, among others, electrical, mechanical, andchemical stimuli (see, e.g., Le Bars et al., 2001, PharmacologicalReviews 53(4):597-652). The stimuli may be short in duration, and can beapplied with variable frequency to test responses to acute pain.Eliciting longer duration pain typically involves intradermal,intraperitoneal, intra-arterial, or intradental administration ofirritating chemical agents, such as formalin and phenylbenzoquinone.

For purposes of treatment, the compound can be administered locally tothe pain site, or administered systemically to a subject in an amounteffective to reduce or eliminate the pain. In other embodiments, thecompounds can be administered prophylactically to prevent the occurrenceor recurrence of pain in the subject. The compounds can be administeredone more times a day until sufficient relief or reduction in pain isobtained by the subject.

5.6 Pharmaceutical Compositions and Administration

When used for treatment of various disorders or conditions, the phorbolcompounds can be administered singly, as mixtures of one or more activecompounds or in a mixture or combination with other agents useful fortreating such diseases and/or symptoms associated with such diseases.The active compounds can be administered per se or as pharmaceuticalcompositions. For pharmaceutical compositions, the phorbol compounds canbe formulated with a pharmaceutically acceptable vehicle. A“pharmaceutically acceptable vehicle” refers to a diluent, adjuvant,excipient or carrier with which the compound is administered.

The composition can be made into a pharmaceutical formulation that iscompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (topical),transmucosal, rectal and oral administration. Solutions or suspensionsused for parenteral, intradermal, or subcutaneous application caninclude the following components: a sterile diluent such as water forinjection, saline solution, fixed oils, polyethylene glycols, glycerine,propylene glycol or other synthetic solvents; antibacterial agents suchas benzyl alcohol or methyl parabens; antioxidants such as ascorbic acidor sodium bisulfite; chelating agents such as ethylenediaminetetraaceticacid; buffers such as acetates, citrates or phosphates; and agents forthe adjustment of tonicity such as sodium chloride or dextrose. The pHcan be adjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

The pharmaceutical formulations can be manufactured in manners generallyknown for preparing pharmaceutical compositions, e.g., usingconventional techniques such as mixing, dissolving, granulating,dragee-making, levigating, emulsifying, encapsulating, entrapping orlyophilizing. Pharmaceutical formulations can be formulated in aconventional manner using one or more physiologically acceptablecarriers, which can be selected from excipients and auxiliaries thatfacilitate processing of the active compounds into preparations whichcan be used pharmaceutically.

A variety of pharmaceutical forms can be employed. Thus, if a solidcarrier is used, the preparation can be tableted, placed in a hardgelatin capsule in powder or pellet form, or in the form of a troche orlozenge. The amount of solid carrier can vary, but generally will befrom about 25 mg to about 1 gm. If a liquid carrier is used, thepreparation will be in the form of syrup, emulsion, soft gelatincapsule, sterile injectable solution or suspension in an ampoule or vialor non-aqueous liquid suspension.

In some embodiments, to obtain a stable water-soluble dose form, apharmaceutically acceptable salt of compound can be dissolved in anaqueous solution of an organic or inorganic acid, such as 0.3M solutionof succinic acid or citric acid. If a soluble salt form is notavailable, the agent can be dissolved in a suitable co-solvent orcombinations of co-solvents. Examples of suitable co-solvents include,but are not limited to, alcohol, propylene glycol, polyethylene glycol300, polysorbate 80, glycerin and the like in concentrations rangingfrom about 0% to about 60% of the total volume.

In other embodiments, the pharmaceutical formulation can also be in theform of a solution of a salt form of the active ingredient in anappropriate aqueous vehicle such as water or isotonic saline or dextrosesolution.

Formulation can be based upon the route of administration chosen. Forinjection, the compounds can be formulated into aqueous solutions,preferably in physiologically compatible buffers such as Hanks'solution, Ringer's solution, or physiological saline buffer. Fortransmucosal administration, penetrants appropriate to the barrier to bepermeated are used in the formulation. Such penetrants are generallyknown in the art.

For oral administration, the compounds can be formulated by combiningwith pharmaceutically acceptable carriers known in the art. Suchcarriers enable the compounds to be formulated as tablets, pills,dragees, capsules, liquids, gels, syrups, slurries, suspensions and thelike, for oral ingestion by a patient to be treated. Pharmaceuticalpreparations for oral use can be obtained using a solid excipient inadmixture with the active ingredient (compound), optionally grinding theresulting mixture, and processing the mixture of granules after addingsuitable auxiliaries, if desired, to obtain tablets or dragee cores.Suitable excipients include: fillers such as sugars, including lactose,sucrose, mannitol, and sorbitol; cellulose preparations, for example,maize starch, wheat starch, rice starch, potato starch, gelatin, gum,methyl cellulose, hydroxypropylmethyl-cellulose, sodiumcarboxymethylcellulose; or polyvinylpyrrolidone (PVP). If desired,disintegrating agents can be added, such as crosslinked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodiumalginate.

Dragee cores can be provided with suitable coatings. For this purpose,concentrated sugar solutions can be used, which can optionally comprisegum horoi, polyvinyl pyrrolidone, Carbopol gel, polyethylene glycol,and/or titanium dioxide, lacquer solutions, and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments can be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compounds and agents.

Pharmaceutical forms which can be used orally include push-fit capsulesmade of gelatin, as well as soft, sealed capsules made of gelatin and aplasticizer, such as glycerol or sorbitol. The push-fit capsules cancomprise the active ingredients in admixture with fillers such aslactose, binders such as starches, and/or lubricants such as talc ormagnesium stearate, and, optionally, stabilizers. In soft capsules, theactive agents can be dissolved or suspended in suitable liquids, such asfatty oils, liquid paraffin, or liquid polyethylene glycols. Inaddition, stabilizers can be added. All formulations for oraladministration should be in dosages suitable for such administration.For buccal administration, the formulations can take the form of tabletsor lozenges formulated in a conventional manner.

Oral formulations generally include an inert diluent or an ediblecarrier. Oral formulations can also be prepared using a fluid carrierfor use as a mouthwash, wherein the compound in the fluid carrier isapplied orally and swished and expectorated or swallowed.Pharmaceutically compatible binding agents, and/or adjuvant materialscan be included as part of the composition. The tablets, pills,capsules, troches and the like can comprise any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate; aglidant such as colloidal silicon dioxide; a sweetening agent such assucrose or saccharin; or a flavoring agent such as peppermint, methylsalicylate, or orange flavoring. Some embodiments of oral formulationsinclude microcrystalline tablets, gelatin capsules, and the like.

For administration intranasally or by inhalation, the compounds can beconveniently delivered in the form of an aerosol spray presented frompressurized packs or a nebuliser, with the use of a suitable propellant,e.g., dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In thecase of a pressurized aerosol, the dosage unit can be determined byproviding a valve to deliver a metered amount. Capsules and cartridgesof gelatin for use in an inhaler or insufflator and the like can beformulated comprising a powder mix of the compound and a suitable powderbase such as lactose or starch.

In other embodiments, the compounds can be formulated for parenteraladministration by injection, e.g., by bolus injection or continuousinfusion. Formulations for injection can be presented in unit-dosageform, e.g., in ampoules or in multi-dose containers, with an addedpreservative. The parenteral formulations can take such forms assuspensions, solutions or emulsions in oily or aqueous vehicles, and cancomprise formulatory agents such as suspending, stabilizing and/ordispersing agents.

Pharmaceutical formulations suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. Aqueous injection suspensions can comprisesubstances which increase the viscosity of the suspension, such assodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, thesuspension can also comprise suitable stabilizers or agents whichincrease the solubility of the compounds to allow for the preparation ofhighly concentrated solutions. Additionally, suspensions of the activeagents can be prepared as appropriate oily injection suspensions.Suitable lipophilic solvents or vehicles include fatty oils such assesame oil, or synthetic fatty acid esters, such as ethyl oleate ortriglycerides, or liposomes.

For intravenous administration, suitable carriers include physiologicalsaline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, N.J.) orphosphate buffered saline (PBS). The composition should be sterile andbe fluid to the extent that easy syringability exists. It should bestable under the conditions of manufacture and storage and must bepreserved against the contaminating action of microorganisms such asbacteria and fungi. The carrier can be a solvent or dispersion mediumcomprising, for example, water, ethanol, polyol (for example, glycerol,propylene glycol, and liquid horoidsene glycol, and the like), andsuitable mixtures thereof. The proper fluidity can be maintained, forexample, by the use of a coating such as lecithin, by the maintenance ofthe required particle size in the case of dispersion and by the use ofsurfactants. Prevention of microbial growth can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in theformulation. Prolonged absorption of the injectable compositions can bebrought about by including in the formulation an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating atherapeutically effective amount of at least one compound disclosedherein in an appropriate solvent with one or a combination ofingredients enumerated above, as required, followed by filteredsterilization. Generally, dispersions can be prepared by incorporatingthe compound into a sterile vehicle that comprises a basic dispersionmedium and the required other ingredients from those enumerated above.In the case of sterile powders for the preparation of sterile injectablesolutions, suitable methods of preparation include vacuum drying andfreeze-drying, which yields a powder of the active compound plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

Systemic administration can also be by transmucosal or transdermalmeans. In some embodiments, penetrants appropriate to the barrier to bepermeated are used in the formulation. Such penetrants are generallyknown in the art, and include, but are not limited to, detergents, bilesalts, and fusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds can be formulated intoointments, salves, gels, foams, powders, sprays, aerosols or creams asgenerally known in the art.

For example, for topical formulations, pharmaceutically acceptableexcipients or cosmetically acceptable carriers and additives includesolvents, emollients, humectants, preservatives, emulsifiers, and pHagents. Suitable solvents include ethanol, acetone, glycols,polyurethanes, and others known in the art. Suitable emollients includepetrolatum, mineral oil, propylene glycol dicaprylate, lower fatty acidesters, lower alkyl ethers of propylene glycol, cetyl alcohol,cetostearyl alcohol, stearyl alcohol, stearic acid, wax, and othersknown in the art. Suitable humectants include, among others, glycerinand sorbitol. Suitable emulsifiers include, among others, glycerylmonostearate, glyceryl monoleate, stearic acid, polyoxyethylene cetylether, polyoxyethylene cetostearyl ether, polyoxyethylene stearyl ether,polyethylene glycol stearate, and propylene glycol stearate. Suitable pHagents include, among others, hydrochloric acid, phosphoric acid,diethanolamine, triethanolamine, sodium hydroxide, monobasic sodiumphosphate, dibasic sodium phosphate. Suitable preservatives includebenzyl alcohol, sodium benzoate, parabens, and others known in the art.

For administration to the eye, the compounds herein can be delivered ina pharmaceutically acceptable ophthalmic vehicle such that the compoundis maintained in contact with the ocular surface for a sufficient timeperiod to allow the compound to penetrate the corneal and internalregions of the eye, including, for example, the anterior chamber,posterior chamber, vitreous body, aqueous humor, vitreous humor, cornea,iris/cilary, lens, retina and sclera. The pharmaceutically acceptableophthalmic vehicle can be an ointment or an encapsulating material.Compounds can also be injected directly into the vitreous and aqueoushumor.

In various embodiments, compounds can be in powder form forreconstitution with a suitable vehicle, e.g., sterile pyrogen-freewater, before use. The compounds can also be formulated in rectalcompositions such as suppositories or retention enemas, e.g., comprisingconventional suppository bases such as cocoa butter or other glycerides.

In addition to the formulations described above, compounds can also beformulated as a depot preparation. Such long-acting formulations can beadministered by implantation (e.g., subcutaneously or intramuscularly)or by intramuscular injection. Thus, for example, the compounds can beformulated with suitable polymeric or hydrophobic materials (e.g., anemulsion in an acceptable oil) or ion-exchange resins, or as sparinglysoluble derivatives, for example, as a sparingly soluble salt.

In some embodiments, a pharmaceutical carrier for hydrophobic compoundsis a cosolvent system comprising benzyl alcohol, a nonpolar surfactant,a water-miscible organic polymer, and an aqueous phase. The cosolventsystem can be a VPD co-solvent system. VPD is a solution of 3% w/vbenzyl alcohol, 8% w/v of the nonpolar surfactant polysorbate 80, and65% w/v polyethylene glycol 300, made up to volume in absolute ethanol.The VPD co-solvent system (VPD:5W) comprises VPD diluted 1:1 with a 5%dextrose in water solution. This co-solvent system dissolves hydrophobiccompounds well, and itself produces low toxicity upon systemicadministration. In various embodiments, he proportions of a co-solventsystem can be varied considerably without destroying its solubility andtoxicity characteristics. Furthermore, the identity of the co-solventcomponents can be varied. For example, other low-toxicity nonpolarsurfactants can be used instead of polysorbate 80; the fraction size ofpolyethylene glycol can be varied; other biocompatible polymers canreplace polyethylene glycol, e.g. polyvinyl pyrrolidone; and othersugars or polysaccharides can be substituted for dextrose.

In some embodiments, other delivery systems for hydrophobicpharmaceutical formulations can be employed. Liposomes and emulsions areknown examples of delivery vehicles or carriers for hydrophobic drugsand cosmetics. Certain organic solvents such as dimethylsulfoxide alsocan be employed, although usually at the cost of greater toxicity.Additionally, the compounds can be delivered using a sustained-releasesystem, such as semipermeable matrices of solid hydrophobic polymerscomprising the therapeutic agent. Various sustained-release materialshave been established and are known by those skilled in the art.Sustained-release capsules can, depending on their chemical nature,release the compounds for a few weeks up to over 100 days.

The pharmaceutical formulations also can comprise suitable solid- orgel-phase carriers or excipients. Examples of such carriers orexcipients include calcium carbonate, calcium phosphate, sugars,starches, cellulose derivatives, gelatin, and polymers such aspolyethylene glycols.

In some embodiments, the compounds are prepared with carriers that willprotect the compound against rapid elimination from the body, such as acontrolled release formulation, including implants and microencapsulateddelivery systems. Biodegradable, biocompatible polymers can be used,such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid,collagen, polyorthoesters, and polylactic acid. Methods for preparationof such formulations will be apparent to those skilled in the art. Thematerials can also be obtained commercially from Alza Corporation andNova Pharmaceuticals, Inc. Liposomal suspensions can also be used aspharmaceutically or cosmetically acceptable carriers. These can beprepared according to methods known to those skilled in the art, forexample, as described in U.S. Pat. No. 4,522,811.

It is generally advantageous to formulate oral or parenteralformulations in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit comprising a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier.

The specification for the dosage unit forms of the compounds aredictated by and directly dependent on the unique characteristics of theactive compound and the particular therapeutic effect to be achieved,and the limitations inherent in the art of compounding such an activecompound for the treatment of individuals.

5.7 Dosages

The active compound(s) or compositions thereof can generally be used inan amount effective to treat or prevent the particular disease beingtreated. The compound(s) can be administered therapeutically to achievetherapeutic benefit or prophylactically to achieve prophylactic benefit.By “therapeutic benefit” is meant eradication or amelioration of theunderlying disorder being treated, e.g., eradication or amelioration ofthe HIV infection, neoplastic disease, inflammation, and/or eradicationor amelioration of one or more of the symptoms associated with theunderlying disorder such that the patient reports an improvement infeeling or condition, notwithstanding that the patient can still beafflicted with the underlying disorder. For example, administration ofan active compound to a subject suffering from HIV infection providestherapeutic benefit not only when the underlying HIV infection iseradicated or ameliorated, but also when the patient reports a decreasein the severity or duration of the symptoms associated with HIV.Therapeutic benefit also includes halting or slowing the progression ofthe disease, regardless of whether improvement is realized.

For prophylactic administration, the active compound can be administeredto a patient at risk of developing the specified disorder or disease.For example, if a subject is suspected of being infected with HIV, theactive compound can be administered to prevent infection before testssensitive enough to detect presence of the virus can be used. Activecompounds can also be administered prophylactically to healthyindividuals who are at high risk of being infected by HIV (e.g.,high-risk sexual activity, illicit intravenous drug abusers, HIV healthworkers, etc.).

The amount of active compound(s) administered will take intoconsideration a variety of factors, including, for example, theparticular indication being treated, the mode of administration, whetherthe desired benefit is prophylactic or therapeutic, the severity of theindication being treated, the age, weight and sex of the patient, andthe bioavailability of the particular active compound. Determination ofan effective dosage is well within the capabilities of those skilled inthe art.

Initial dosages can be estimated initially from in vitro assays. Forexample, an initial dosage for use in animals can be formulated toachieve a circulating blood or serum concentration of active compoundthat inhibits about 50% of HIV viral replication as measured in an invitro assay. In other embodiments, an initial dosage for use in animalscan be formulated to achieve a circulating blood or serum concentrationof active compound that is equal to or greater than the IC₅₀ of a invivo model of HIV infection. Such in vivo models can be primate modelsfor HIV infection (see, e.g., Vodros et al., 2004, Acta MicrobiolImmunol Hung. 51(1-2):1-29).

Calculating dosages to achieve such circulating blood or serumconcentrations, taking into account the bioavailability of theparticular active compound, is well within the capabilities of skilledartisans. For guidance, the reader is referred to Fingl and Woodbury,“General Principles,” In: The Pharmaceutical Basis of Therapeutics,Chapter 1, pp. 1-46, 1975, and the references cited therein.

In some embodiments, the active compound(s) will provide therapeutic orprophylactic benefit without causing substantial toxicity. Toxicity ofthe active compound(s) can be determined using standard pharmaceuticalprocedures. The dose ratio between toxic and therapeutic (orprophylactic) effect is the therapeutic index. Active compound(s) thatexhibit high therapeutic indices are preferred.

Dosage amounts can typically be in the range of from about 0.1 mg/kg/dayto about 1.0 mg/kg/day, 1.5 mg/kg/day, 2.0 mg/kg/day, 2.5 mg/kg/day or10.0 mg/kg/day, but can be higher or lower, depending upon, among otherfactors, the activity of the compound, its bioavailability, the mode ofadministration and various factors discussed above. Dosage amount andinterval can be adjusted individually to provide plasma levels of theactive compound(s) that are sufficient to maintain therapeutic orprophylactic effect. In cases of local administration or selectiveuptake, such as local topical administration, the effective localconcentration of active compound(s) may not be related to plasmaconcentration. Optimize effective local dosages are well within theskill of those in the art.

In some embodiments, the compound(s) can be administered once per day, afew or several times per day, or even multiple times per day, dependingupon, among other things, the indication being treated and the judgmentof the prescribing physician.

5.8 Kits

The present disclosure also provides the compounds and compositionsdisclosed herein in the form of a kit or packaged formulation. A kit orpackaged formulation can include one or more dosages of the prodrugphorbol compound, or salts and hydrates thereof. In some embodiments,the kit further comprises one or more therapeutic agents usedadjunctively with the phorbol compounds in the treatment of a disorderor disease condition. For instance, the additional therapeutic agent canbe an anti-retroviral agent provided in dosage forms, such as pills orcapsules packaged into dispensers or blister packs, together withinstructions for simultaneous or sequential administration of thetherapeutic agents. In other embodiments, the package can contain thephorbol compound along with a pharmaceutical carrier combined in theform of a powder for mixing in an aqueous solution, which can beingested by a subject. Another example of packaged drug is a preloadedpressure syringe so that the compositions can be delivered orally orintravenously in measured doses. The package or kit also includesappropriate instructions, which encompasses diagrams, recordings (e.g.,audio, video, compact disc), and computer programs providing directionsfor use of the mono- or combination therapy.

6. EXAMPLES 6.1 Example 1 Synthesis of Prostratin Succinate (SA-101B)

Prostratin (1390 mg, 1 mmol) and succinic anhydride (120 mg, 1.2 mmol)were dissolved in dichloromethane (20 ml). DMAP (dimethylaminopyridine)at 50 mg was added under stirring and then stirred at room temperaturefor overnight. The solvent was evaporated and the residue subjected tochromatography on silica gel column (50 mg) with gradient of petroleumether (60-90° C.) and acetone as eluent to give a fraction of pureprostratin succinate (Scheme (1), product 1) and another fractioncontaining a mixture of prostratin and prostratin succinate.

EIMS m/z: 490 (M⁺, 2%), 472 (M⁺-H₂O, 25%), 312 (M+-H₂O—HOAc-HOSuc,100%), 294 (M⁺-2H₂O—HOAc-HOSuc, 94%). ¹H-NMR 6 (DMCO): 0.81 (3H, d,J=6.0, H-18), 0.84 (1H, d, J=5.4, H-14), 0.98 (3H, S, H-17), 1.10 (3H,s, H-16), 1.48 (1H, dd, J=8.5, 13.2, H-12a), 1.63 (3H, dd, J=1.1; 2.8,H-19), 1.98 (3H, s, H-22), 1.98 (1H, m, H-12b), 2.32 (1H, d, J=19.5,H-11), 2.48 (1H, br. H-5), 2.48 (4H, br. H-24, 25), 3.01 (1H, t, J=5.2,H-8), 3.11 (1H, t, J=2.5, H-10), 4.39, 4.47 (2H, AB, J=12.1, H-20), 5.65(1H, d, J=3.9, H-7), 6.98 (1H, d, J=7.4, H-1), 8.15 (1H, d, J=7.4,H-26).

6.2 Example 2 Prostratin Succinate Sodium (SA-101NB)

Prostratin succinate (2340 mg, 0.69 mmol) was added proportionately to awater solution of sodium bicarbonate (58 mg, 0.69 mmol) under stirringat room temperature and then continuously stirred for another two hours.The solution was frozen and subjected to lyophilization to yield anoffwhite powder, which is the sodium salt of product (1) of Scheme (1).

¹H-NMR δ (D₂O): 0.74 (3H, d, J=6.6, H-18), 0.82 (1H, d, J=6.2, H-14),0.86 (3H, s, H-17), 0.96 (3H, s, H-16), 1.38 (1H, dd, J=2.5; 3.8,H-12a), 1.58 (3H, s, H-19), 1.69 (1H, m, H-11), 1.92 (3H, s, H-22), 2.03(1H, dd, J=7.2; 15.2, H-12b), 2.50 (3H, m, H-5, 24), 2.37 (2H, m, H-25),2.72 (1H, t, J=5.4, H-8), 3.04 (1H, s, H-10), 4.35 (2H, brs, H-20), 5.58(1H, d, J=4.2, H-7), 7.56 (1H, s, H-1).

¹³C-NMR δc (D₂O): 162.4 (C-1), 135.0 (C-2), 211.5 (C-3), 74.4 (C-4),37.2 (C-5), 135.7 (C-6), 132.8 (C-7), 39.0 (C-8), 77.3 (C-9), 56.4(C-10), 36.2 (C-11), 31.6 (C-12), 62.8 (C-13), 30.9 (C-14), 24.1 (C-15),22.1 (C-16), 15.0 (C-17), 18.1 (C-18), 9.7 (C-19), 70.2 (C-20), 176.0(C-21), 20.8 (C-22), 176.2 (C-23), 30.7 (C-24), 32.3 (C-25), 181.1(C-26).

6.3 Example 3 α-Aminosuccinate of Prostratin

2-Formamido-succinic anhydride (28 mg, 0.1 mmol) in 2 ml ofdichloromethane was treated with 1,3-dicyclohexylcarbodiimide (DCC) (2mmol) under stirring at room temperature for 2 hrs. A solution ofprostratin (139 mg, 0.1 mmol) and DMAP (24 mg, 0.2 mmol) in CH₂Cl₂ wasadded and stirred under nitrogen over night. The precipitate was removedand the solvent evaporated to dryness. Compound was purified bypreparative TLC to give pure product (Scheme (1), product 2).

6.4 Example 4 Solubility of Prostratin and SA-101B

Solubility of prostratin and SA-101B was measured in pH controlledsolutions and in various representative formulations, in particular,intravenous formulations, where solubility is often a limiting factor.

With reference to the data presented in Table 1, SA-101B had a bettersolubility than prostratin in all media.

TABLE 1 Solubility (in mg/mL) Saline 5% (0.9% Compound # pH 1 pH 2 pH 5pH 7.5 pH 9 Dextrose NaCl) SA-101A 0.37 0.37 0.45 0.42 0.39 0.33 0.34SA-101B 1.21 4.46 7.72 11.20 12.08 >10 >10

6.5 Example 5 Antiviral Activity of SA-101B Against HIV-1 in HumanMacrophages

SA-101A and SA-101B were tested for antiviral activity against HIV-1virus in peripheral blood monocytes/macrophages. Monocytes/macrophageswere derived from normal HIV-1 negative donors, and cultured underconditions which promote cell survival and HIV replication. Fresh humanblood was obtained commercially from Biological Specialty Corporation(Colmar, Pa.). The low passage, monocytropic, clinical isolate HIV-1Ba-L(Subtype B, R5) was obtained from the NIAID AIDS Research and ReferenceReagent Program. A pre-titered aliquot of HIV 1Ba-L was removed from thefreezer (−80° C.) and thawed rapidly to room temperature in a biologicalsafety cabinet immediately before use. Phytohemagglutinin (PHA-P) wasobtained from Sigma (St. Louis, Mo.) and recombinant IL-2 was obtainedfrom R&D Systems Inc. (Minneapolis, Minn.).

Compound Preparation:

Compounds SA-101A and SA-101B were in powder form and solubilized inDMSO prior to use. AZT (Sigma), solubilized in sterile dH₂O, was used asthe positive antiviral control. The compounds were tested at a 100 μg/mLhigh-test concentration; AZT was tested at a 1,000 ηM high-testconcentration. Compound SA-101B was tested in the presence of 40% humanAB serum.

Monocyte Isolation and Culture.

Peripheral blood monocytes were isolated from screened donorssero-negative for HIV and HBV. Cells were pelleted by low speedcentrifugation and re-suspended in PBS to remove contaminatingplatelets. The leukophoresed blood was then diluted with Dulbecco'sphosphate buffered saline (PBS) and layered over of LymphocyteSeparation Medium (LSM; Cellgro® by Mediatech, Inc.; density1.078+/−0.002 g/ml; Cat. #85-072-CL) in a 50 mL centrifuge tube and thencentrifuged. Banded PBMCs were gently aspirated from the interface andsubsequently washed with PBS by low speed centrifugation. The cells werediluted to 4×10⁶ cells per mL in DMEM without phenol red supplementedwith 10% heat inactivated human pooled AB serum, 2 mM L-glutamine, 100U/mL penicillin and 100 μg/mL streptomycin. Monocytes/macrophages wereallowed to adhere to the interior 60 wells (100 μL/well) of a 96 wellflat bottomed plate for 2 to 18 hours at 37° C., 5% CO₂. The exteriorwells were filled with 200 μL of sterile DPBS to serve as a humiditybarrier. Following adherence of cells, the cultures were washed withsterile DPBS to remove non-adherent cells (lymphocytes and contaminatingRBCs). Subsequently, 200 μL of RPMI 1640 supplemented with 15% FBS, 2 mML-glutamine, 100 U/mL penicillin and 100 μg/mL streptomycin was added tothe wells. The plates were incubated at 37° C. in a humidified incubatorwith 5% CO₂. Culture medium was replaced once per week until use.Culture plates were used for anti-HIV evaluations between days 6 and 14of incubation following initial isolation of the cells.

Anti-HIV Efficacy Evaluation in Human Monocyte/Macrophages.

Following 6 to 14 days in culture, the monocyte/macrophages cultureswere washed 3 times to remove any non-adherent cells, and seriallydiluted test compounds were added followed by the addition of apre-titered amount of HIV. Cultures were washed a final time by mediaremoval 24 h post infection, fresh compound added and the culturescontinued for an additional six days. Assays were performed using astandardized microtiter plate format developed by the Infectious DiseaseResearch Department of Southern Research Institute. Each plate containsvirus/cell control wells (cells plus virus), experimental wells (drugplus cells plus virus) and compound control wells (drug plus mediawithout cells, necessary for MTS monitoring of cytotoxicity). Since HIVis not cytopathic to monocytes/macrophages, this allows the use of thesame assay plate for both antiviral activity and cytotoxicitymeasurements. At assay termination, virus replication was measured bycollecting cell-free supernatant samples, which were analyzed for HIVp24 antigen content using a commercially available p24 ELISA assay(Coulter). Following removal of supernatant samples, compoundcytotoxicity was measured by addition of MTS to the plates fordetermination of cell viability. Wells were also examinedmicroscopically and any abnormalities noted. The HIV reversetranscriptase inhibitor AZT was used as a positive control compound andrun in parallel with each determination.

p24 Antigen ELISA.

ELISA kits were purchased from Coulter Electronics, and detection ofsupernatant or cell-associated p24 antigen is performed according to themanufacturer's instructions. All p24 determinations were performedfollowing serial dilution of the samples to ensure absorbances in thelinear range of the standard p24 antigen curve. The standard curve isproduced using manufacturer-supplied standards and instructions. Datawere obtained by spectrophotometric analysis at 450 ηm using a MolecularDevices Vmax or SpectraMaxPlus plate reader. Final concentrations werecalculated from the optical density values using the Molecular DevicesSoftMax Pro software package and expressed in pg/ml p24 antigen.

MTS Staining for Macrophage Viability to Measure Cytotoxicity.

At assay termination, the assay plates were stained with the solubletetrazolium-based dye MTS (CellTiter Reagent, Promega) to determine cellviability and quantify compound cytotoxicity. MTS is metabolized by themitochondrial enzymes of metabolically active cells to yield a solubleformazan product, allowing the rapid quantitative analysis of cellviability and compound cytotoxicity. At termination of the assay, 20 μLof MTS reagent was added per well and the plates incubated for 4 hrs at37° C. Adhesive plate sealers were used in place of the lids, the sealedplate was inverted several times to mix the soluble formazan product.Developed plates were read spectrophotometrically at 490/650 ηm with aMolecular Devices Vmax plate reader.

Results.

Compounds SA-101A and SA-101B were evaluated for anti-HIV activity inhuman macrophage cultures using HIV-1 isolate Ba-L. SA-101B was used inthe presence of 40% human AB serum. The results are summarized in Table2.

Both compounds demonstrated activity against HIV-1Ba-L with IC₅₀ valuesof 0.11 μg/mL for SA-101A and 0.28 μg/mL for SA-101B. Both compoundsalso displayed some cytotoxicity with TC₅₀ values of 17.3 μg/mL and 21.0μg/mL respectively.

The control compound AZT, used in the macrophage assay in parallel withthe test compounds, yielded IC₅₀ values that fell within the acceptableranges normally observed when performing antiviral assays. Macroscopicobservation of the cells in each well of the microtiter plate confirmedthe cytotoxicity results obtained following staining of the cells withthe MTS metabolic dye.

TABLE 2 SA-101B vs. HIV-1_(Ba-L) in Human Macrophages TherapeuticCompound IC₉₀ IC₅₀ TC₅₀ Index Prostratin 0.28 0.11 17.3 156 μg/mL μg/mLμg/mL AZT 123 9.30 >1,000 >108 nM nM nM SA-101B 7.29 0.28 21.0 74.8 40%Human μg/mL μg/mL μg/mL AB serum

6.6 Example 6 Antiviral Activity of SA-101A and SA-101B Against HIV-1 inHuman PBMCs

Compounds SA-101A and SA-101B were tested for antiviral activity againstHIV-1 in peripheral blood mononuclear cells (PBMC) acutely infected withthe HIV-1 isolates. PBMCs were derived from normal HIV-1 negativedonors, and cultured under conditions which promote cell survival andHIV replication. Fresh human blood was obtained commercially fromBiological Specialty Corporation (Colmar, Pa.). The low passage clinicalisolates HIV-1Ba-L, HIVIIIB, and SIVmac251 were from the NIAID AIDSResearch and Reference Reagent Program. Immediately before use,pre-titered aliquot of the viruses were removed from storage in afreezer (−80° C.) and thawed rapidly to room temperature in a biologicalsafety cabinet. Phytohemagglutinin (PHA-P) was obtained from Sigma (St.Louis, Mo.) and recombinant IL-2 was obtained from R&D Systems Inc.(Minneapolis, Minn.). Antiviral activity was tested from a high-testconcentration of 100 μM or 100 μg/ml with nine ½-log serial dilutions ofthe compound to derive applicable IC₅₀ (concentration inhibition virusreplication by 50%), IC₉₀ (concentration inhibition virus replication by90%), TC₅₀ (concentration decreasing cell viability by 50%) and TI(therapeutic index: TC₅₀/IC₅₀) values.

Preparation of PMBC.

Fresh human PBMCs, seronegative for HIV and HBV, were isolated fromscreened donors (Interstate Blood Bank, Inc. Memphis, Tenn.). Cells werepelleted/washed 2-3 times by low speed centrifugation and resuspensdedin PBS to remove contaminating platelets. The leukophoresed blood wasthen diluted 1:1 with Dulbecco's Phosphate Buffered Saline (DPBS) andlayered over 14 mL of Lymphocyte Separation Medium (LSM; Cellgro® byMediatech, Inc.; density 1.078+/−0.002 g/ml; Cat. #85-072-CL) in a 50 mLcentrifuge tube and then centrifuged for 30 minutes at 600×g. BandedPBMCs were gently aspirated from the resulting interface andsubsequently washed 2× with PBS by low speed centrifugation. After thefinal wash, cells were enumerated by trypan blue exclusion andre-suspended at 1×10⁷ cells/mL in RPMI 1640 supplemented with 15% FetalBovine Serum (FBS), and 2 mM L-glutamine, 4 μg/mL Phytohemagglutinin(PHA-P, Sigma). The cells were allowed to incubate for 48-72 hours at37° C. After incubation, PBMCs were centrifuged and re-suspended in RPMI1640 with 15% FBS, 2 mM L-glutamine, 100 U/mL penicillin, 100 μg/mLstreptomycin, 10 μg/mL gentamycin, and 20 U/mL recombinant human IL-2(R&D Systems, Inc). IL-2 is included in the culture medium to maintainthe cell division initiated by the PHA mitogenic stimulation. PBMCs weremaintained in this medium at a concentration of 1−2×10⁶ cells/mL withbiweekly medium changes until used in the assay protocol. Cells werekept in culture for a maximum of two weeks before being deemed too oldfor use in assays and discarded. Monocytes were depleted from theculture as the result of adherence to the tissue culture flask.

PMBC Assay.

For the standard PBMC assay, PHA-P stimulated cells from at least twonormal donors were pooled (mixed together), diluted in fresh medium to afinal concentration of 1×10⁶ cells/mL, and plated in the interior wellsof a 96 well round bottom microplate at 50 μL/well (5×10⁴ cells/well) ina standard format developed by the Infectious Disease Researchdepartment of Southern Research Institute. Pooling (mixing) ofmononuclear cells from more than one donor is used to minimize thevariability observed between individual donors, which results fromquantitative and qualitative differences in HIV infection and overallresponse to the PHA and IL-2 of primary lymphocyte populations. Eachplate contains virus/cell control wells (cells plus virus), experimentalwells (drug plus cells plus virus) and compound control wells (drug plusmedia without cells, necessary for MTS monitoring of cytotoxicity).Since HIV-1 is not cytopathic to PBMCs, this allows the use of the sameassay plate for both antiviral activity and cytotoxicity measurements.Test drug dilutions were prepared at a 2× concentration in microtitertubes and 100 μL of each concentration was placed in appropriate wellsusing the standard format. 50 μL of a predetermined dilution of virusstock was placed in each test well (final MOI˜0.1). The PBMC cultureswere maintained for seven days following infection at 37° C., 5% CO₂.After this period, cell-free supernatant samples were collected foranalysis of reverse transcriptase activity and/or HIV p24 content.Following removal of supernatant samples, compound cytotoxicity wasmeasured by addition of MTS to the plates for determination of cellviability. Wells were also examined microscopically and anyabnormalities were noted.

Reverse Transcription Activity Assay.

Reverse transcriptases activity measurements used the protocol describedin Buckheit et al., 1991, AIDS Research and Human Retroviruses7:295-302. Specifically, tritiated thymidine triphosphate (³H-TTP, 80Ci/mmol, NEN) was received in 1:1 dH₂O:Ethanol at 1 mCi/mL. PolyrA:oligo dT template:primer (Pharmacia) was prepared as a stock solutionby combining 150 μL poly rA (20 mg/mL) with 0.5 mL oligo dT (20units/mL) and 5.35 mL sterile dH₂O followed by aliquoting (1.0 mL) andstorage at −20° C. The RT reaction buffer was prepared fresh on a dailybasis and consisted of 125 μL 1.0 M EGTA, 125 μL dH₂O, 125 μL 20% TritonX100, 50 μL 1.0 M Tris (pH 7.4), 50 μL 1.0 M DTT, and 40 μL 1.0 M MgCl₂.The final reaction mixture was prepared by combining 1 part ³H-TTP, 4parts dH₂O, 2.5 parts poly rA:oligo dT stock and 2.5 parts reactionbuffer. Ten microliters of this reaction mixture was placed in a roundbottom microtiter plate and 15 μL of virus containing supernatant wasadded and mixed. The plate was incubated at 37° C. for 60 minutes.Following incubation, the reaction volume was spotted onto DE81filter-mats (Wallac), washed 5 times for 5 minutes each in a 5% sodiumphosphate buffer or 2×SSC (Life Technologies). Next they were washed 2times for 1 minute each in distilled water, 2 times for 1 minute each in70% ethanol, and then dried. Incorporated radioactivity (counts perminute, CPM) was quantified using standard liquid scintillationtechniques.

p24 Antigen ELISA.

ELISA kits are purchased from Coulter Electronics, and detection ofsupernatant p24 antigen is performed according to the manufacturer'sinstructions. All p24 determinations are performed following serialdilution of the samples to ensure absorbances in the linear range of thestandard p24 antigen curve. The standard curve is produced usingmanufacturer-supplied standards and instructions. Data are obtained byspectrophotometric analysis at 450/570 ηm using a Molecular Devices Vmaxor SpectraMaxPlus plate reader. Final concentrations are calculated fromthe optical density values using the Molecular Devices SoftMax Prosoftware package and expressed in pg/ml p24 antigen.

MTS Staining for PBMC Viability to Measure Cytotoxicity.

At assay termination, assay plates were stained with the solubletetrazolium-based dye MTS (CellTiter 96 Reagent, Promega) to determinecell viability and quantify compound toxicity. The mitochondrial enzymesof metabolically active cells metabolize MTS to yield a soluble formazanproduct. This allows the rapid quantitative analysis of cell viabilityand compound cytotoxicity. The MTS is a stable solution that does notrequire preparation before use. At termination of the assay, 20 μL ofMTS reagent was added per well. The microtiter plates were thenincubated 4 6 hrs at 37° C. The incubation intervals were chosen basedon empirically determined times for optimal dye reduction. Adhesiveplate sealers were used in place of the lids, the sealed plate wasinverted several times to mix the soluble formazan product and the platewas read spectrophotometrically at 490/650 ηm with a Molecular DevicesVmax or SpectraMaxPlus plate reader.

Data Analysis.

Using an in-house computer program, IC₅₀ (50%, inhibition of virusreplication), IC₉₀ (90%, inhibition of virus replication), IC₉₅ (95%,inhibition of virus replication), TC₅₀ (50% reduction in cellviability), TC₉₀ (90% reduction in cell viability), TC₉₅ (95% reductionin cell viability), and a therapeutic index (TI=IC₅₀/IC₅₀) weredetermined. AZT (nucleoside reverse transcriptase inhibitor) was used asa positive control antiviral compound.

TABLE 3 SA-101A or SA-101B vs. HIV-1_(Ba-L) in Human PBMCs TherapeuticCompound IC₉₀ IC₅₀ TC₅₀ Index SA-101A 0.39 0.05 >20.1 (Pro- μg/mL μg/mLstratin) AZT 499 4.66 >1,000 >215 nM nM nM SA-101B >1.000.0007 >1.00 >1,361 40% μg/mL μg/mL μg/mL Human AB serum AZT 23.90.38 >1,000 >2,625 40% nM nM nM Human AB serum

TABLE 4 SA-101A or SA-101B vs. HIV-1_(IIIB) in Human PBMCs TherapeuticCompound IC₉₀ IC₅₀ TC₅₀ Index SA-101A 0.50 0.09 >1.00 >11.1 (Prostratin)μg/mL μg/mL μg/mL AZT 58.3 4.53 >1,000 >221 nM nM nMSA-101B >1.00 >1.00 >1.00 NA 40% Human AB μg/mL μg/mL μg/mL serum AZT74.3 2.01 >1,000 >499 40% Human AB nM nM nM serum NA = Not achieved

TABLE 5 SA-101A and SA-101B vs. SIVMac251 in Human PBMCs. TherapeuticCompound IC₉₀ IC₅₀ TC₅₀ Index SA-101A 0.90 0.39 >1.00 >2.54 (Prostratin)μg/mL μg/mL μg/mL AZT 474 11.9 >1,000 >84.2 nM nM nMSA-101B >1.00 >1.00 >1.00 NA 40% Human AB μg/mL μg/mL μg/mL serum AZT86.7 6.07 >1,000 >165 40% Human AB nM nM nM serum NA = Not achieved

Results.

SA-101A and SA-101B were evaluated in a PBMC-based assay using the HIV-1isolates Ba-L and IIIB and the SIV isolate mac251. SA-101B was testedagainst all three isolates in the presence of 40% human AB serum.

The preliminary results show that SA-101A demonstrated activity againstall three isolates evaluated in this study with IC₅₀ values ranging from0.05 to 0.39 μg/mL. The therapeutic indices ranged from >2.54 to 20.1.There was no cytotoxicity associated with this compound at theconcentrations evaluated.

The preliminary results also demonstrated activity of SA-101B againstHIV-1Ba-L with an IC₅₀ value of 0.0007 μg/mL and displayed nocytotoxicity at the concentrations evaluated. In the evaluations ofSA-101B against HIV-1IIIB and SIVmac251, the preliminary data suggestthat this compound did not achieve IC₅₀ values in the concentrationstested. Further evaluations of the compounds against the various viralisolates can be warranted under defined conditions that wouldefficiently generate the active form of the phorbol compounds.

The control compound AZT was used in the PBMC assay in parallel with thetest compounds and yielded IC₅₀ values that fell within the acceptableranges normally observed when performing antiviral assays. Macroscopicobservation of the cells in each well of the microtiter plate confirmedthe cytotoxicity results obtained following staining of the cells withthe MTS metabolic dye.

6.7 Example 7 Evaluation of Compounds SA-101A and SA-101B for Anti-HIV-1Efficacy in CEM-SS Cells

Antiviral efficacy of SA-101A and SA-101B compounds were evaluated in anHIV 1 antiviral cytoprotection assay using CEM-SS cells and thelaboratory adapted HIV 1IIIB virus strain.

Compound Preparation.

Compounds SA-101A and SA-101B were prepared in powder form andsolubilized in DMSO and stored at −20° C. AZT (Sigma), solubilized insterile H₂O, was used as a positive antiviral control and was stored at4° C. The compounds were evaluated at a high test concentration of 500μg/mL. AZT was run in parallel at a concentration of 500 nM.

Cell Preparation.

CEM-SS cells were passaged in T-75 flasks prior to use in the antiviralassay. On the day preceding the assay, the cells were split 1:2 toassure they were in an exponential growth phase at the time ofinfection. Total cell and viability quantification was performed using ahemacytometer and trypan blue exclusion. Cell viability was greater than95% for the cells to be utilized in the assay. The cells wereresuspended at 5×10⁴ cells/ml in tissue culture medium and added to thedrug-containing microtiter plates in a volume of 50 μl.

Virus Preparation.

The virus used for these tests was the lymphocytropic virus strain HIV1IIIB. This virus was obtained from the NIH AIDS Research and ReferenceReagent Program and was grown in CEM SS cells for the production ofstock virus pools. For each assay, a pre-titered aliquot of virus wasremoved from the freezer (−80° C.) and allowed to thaw slowly to roomtemperature in a biological safety cabinet. The virus was resuspendedand diluted into tissue culture medium such that the amount of virusadded to each well in a volume of 50 μl was the amount determined togive approximately 90% cell killing at 6 days post-infection. TCID₅₀calculations by endpoint titration in CEM-SS cells indicated that themultiplicity of infection of these assays was approximately 0.01.

MTS Staining for Cell Viability.

At assay termination, the assay plates were stained with the solubletetrazolium-based dye MTS (Cell Titer Reagent Promega) to determine cellviability and quantify compound toxicity. At termination of the assay,20 μL of MTS reagent was added per well. The microtiter plates were thenincubated 46 hrs at 37° C. Adhesive plate sealers were used in place ofthe lids, the sealed plate was inverted several times to mix the solubleformazan product and the plate was read spectrophotometrically at490/650 ηM with a Molecular Devices Vmax plate reader.

Data Analysis.

Using an in-house computer program, IC₅₀ (50%, inhibition of virusreplication), IC₉₅ (95%, inhibition of virus replication), TC₅₀ (50%reduction in cell viability), TC₉₅ (95% reduction in cell viability),and a therapeutic index (TI=TC₅₀/IC₅₀) were determined.

Results.

The results of the assay are summarized in Table 6.

TABLE 6 Activity of Compounds SA-101A and SA-101B Against HIV-1_(IIIB)in CEM-SS Cells Compound IC₅₀ TC₅₀ Therapeutic Index SA-101A 0.086μg/mL >0.50 μg/mL >5.83 SA-101B NA >0.50 μg/mL NA AZT 0.012 μM >0.50μM >41.1 AZT 0.009 μM >0.50 μM >55.1 NA = Not achieved

SA-101A was active in this assay with an IC₅₀ value of 0.086 μg/mL and atherapeutic index of >5.83. This compound demonstrated no cytotoxicityat the concentrations evaluated in this assay.

SA-101B did not achieve an IC₅₀ value and was deemed inactive in thisassay. This compound demonstrated no cytotoxicity at the concentrationsevaluated in the assay.

The control compound AZT was evaluated in parallel with the testcompounds and yielded IC₅₀ values that fell within the acceptable rangesnormally observed when performing antiviral assays. Macroscopicobservation of the cells in each well of the microtiter plate confirmedthe cytotoxicity results obtained following staining of the cells withthe MTS metabolic dye.

6.8 Example 8 HEPG2 2.2.15 Antiviral Assays: Effect on Hepatitis B (HBV)In Vitro

Compounds SA-101A and SA-101B were evaluated for effect on Hepatitis BVirus replications using a cell culture based HBV replication system.

Compound Preparation.

The compounds were prepared in powder form (˜1 mg) and solubilized inDMSO prior to use. Compound 3TC was used as positive antiviral control.

HepG2 2.2.15 Antiviral Assay.

The assay developed is similar to that described ion the literature(Korba et al., 1991, Antiviral Res. 15: 217-228; Korba et al., 1992,Antiviral Res. 19: 55-70) with the exception that viral DNA detectionand quantification have been improved and simplified (Buckwold et al.,2004, Antiviral Res. 61(1): 57-62). Briefly, HepG2-2.2.15 cells areplated in 96-well microtiter plates. Only the interior wells areutilized to reduce “edge effects” observed during cell culture; theexterior wells are filled with complete medium to help minimize sampleevaporation. After 16-24 hours the confluent monolayer of HepG2-2.2.15cells is washed and the medium is replaced with complete mediumcontaining various concentrations of a test compound in triplicate. 3TCis used as the positive control, while media alone is added to cells asa negative control (virus control, VC). Three days later the culturemedium is replaced with fresh medium containing the appropriatelydiluted drug. Six days following the initial administration of the testcompound, the cell culture supernatant is collected, treated withpronase and DNAse and then used in a real-time quantitative TaqMan PCRassay. The PCR-amplified HBV DNA is detected in real-time by monitoringincreases in fluorescence signals that result from the exonucleolyticdegradation of a quenched fluorescent probe molecule that hybridizes tothe amplified HBV DNA. For each PCR amplification, a standard curve issimultaneously generated using dilutions of purified HBV DNA. Antiviralactivity is calculated from the reduction in HBV DNA levels (IC₅₀). Adye uptake assay is then employed to measure cell viability which isused to calculate toxicity (TC₅₀). The therapeutic index (TI) iscalculated as TC₅₀/IC₅₀.

Results.

Compounds SA-101A and SA-101B did not achieve EC₅₀ values in this assayand thus appeared inactive for inhibiting HBV in the particular assayformat. There was no cytotoxicity associated with these compounds at theconcentrations evaluated.

6.9 Example 9 HCV RNA Replicon Antiviral Assays

Compounds SA-101A and SA-101B were evaluated for their effect on HCV RNAreplicons using an in vitro HCV virus replication system.

Compound Preparation.

Compounds were prepared as a powder (˜1 mg) and solubilized in DMSO.IFNα was used as a positive antiviral control.

HCV RNA Replicon System.

The assay for HCV replication employed cell line ET (luc-ubi-neo/ET),which is a human Huh7 hepatoma cell line that contains an HCV RNAreplicon with a stable luciferase (Luc) reporter and three cellculture-adaptive mutations (Pietschmann et al., 2002, J. Virol.76:4008-4021). The HCV RNA replicon contains the 5′ end of HCV (with theHCV Internal Ribosome Entry Site (IRES) and the first few amino acids ofthe HCV core protein) which drives the production of a fireflyluciferase (Luc), ubiquitin (Ubi), and neomycin phosphotransferase(NeoR) fusion protein. Ubiquitin cleavage releases the Luc and NeoRproteins. The EMCV IRES element controls the translation of the HCVstructural proteins NS3-NS5. The NS3 protein cleaves the HCV polyproteinto release the mature NS3, NS4A, NS4B, NS5A and NS5B proteins that arerequired for HCV replication. At the 3′ end of the replicon is theauthentic 3′ NTR of HCV. The Luc reporter is used as an indirect measureof HCV replication. The activity of the Luc reporter is directlyproportional to HCV RNA levels and positive control antiviral compoundsbehave comparably using either Luc or RNA endpoints.

Luciferase Assay.

The ET cell line is grown in Dulbecco's modified essential media (DMEM),10% fetal bovine serum (FBS), 1% penicillin-streptomycin (pen-strep), 1%glutamine, 5 mg/ml G418 in a 5% CO₂ incubator at 37° C. All cell culturereagents are from Gibco Life Technologies. Cells are trypsinized (1%trypsin:EDTA) and plated out at 5×10³ cells/well in white 96-well assayplates (Costar) dedicated to cell number (cytotoxicity) or antiviralactivity (Luc) assessments. Drugs are added at five half-logconcentrations each and the assay is run in DMEM, 5% FBS, 1% pen-strep,1% glutamine. Human interferon alpha-2b (PBL Biolabs, New Brunswick,N.J.) is included in each run as a positive control compound. Cells areprocessed 72 hr post drug addition when the cells are still subconfluent. Compound IC₅₀ and IC₉₀ values (antiviral activity) arederived from plots of HCV RNA levels assessed as HCV RNAreplicon-derived Luc activity versus drug concentration using Steady-Gloassay reagent (Promega), with Luc luminescence read on a 1450 Microbetaliquid scintillation and luminescence counter. Compound TC₅₀ and TC₉₀values (cytotoxicity) are calculated from plots of cell number vs. drugconcentration using data from the CytoTox-ONE cell proliferation assay(Promega); with fluorescence read using an Analyst HT fluorometer. TheCytoTox-ONE assay is an assay of the number of live cells present. Itinvolves lysing cells to release LDH which drives the conversion ofresauzurin to the fluorescent resorufin. Compound selectivity indices(SI=TC/IC) SI₅₀ and SI₉₀ values are calculated from spreadsheets.

Results.

Compound SA-101B was not active in the assay (EC₅₀ was not reached) andit did not display any appreciable cytotoxicity (IC₅₀ was not reached).

Compound SA-101A did not appear to be active or cytotoxic, but appearedto inhibit and later stimulate the cells to grow in a dose-dependentmanner. Studies with lower concentration of the drug can be moreinformative since the lowest concentrations employed in the assayresulted in greater than 50% inhibition of HCV RNA replicon associatedluciferase activity. As such, an EC₅₀ for this compound was notobtainable from the results.

The foregoing descriptions of specific embodiments of the presentdisclosure have been presented for purposes of illustration anddescription. They are not intended to be exhaustive or to limit thescope of the disclosure to the precise forms disclosed, and obviouslymany modifications and variations are possible in light of the aboveteaching.

All patents, patent applications, publications, and references citedherein are expressly incorporated by reference to the same extent as ifeach individual publication or patent application was specifically andindividually indicated to be incorporated by reference.

What is claimed is:
 1. A compound according to structural formula (I)R—X—O—C(O)—R′  (I) including salts and hydrates thereof, wherein: R is aresidue of a phorbol ester, wherein the phorbol ester isingenol-3-acetate, ingenol-3-propionate, ingenol-3-palmitate,ingenol-3-angelate or ingenol-3-benzoate; X is an alkylene chaincontaining from 1 to 12 carbon atoms; R′ is a moiety that is ionizableat a pH in the range of about 2.0-8.0, wherein R′ comprises a carboxylgroup of the formula —(CH₂)_(m)—C(O)OM, m being an integer ranging from1 to 4, or wherein R′ comprises an amino acid selected from—(CH₂)_(n)—CH[(CH₂)_(n)—NH₂]—(CH₂)_(n)—C(O)OM and—(CH₂)_(n)CH(NH₂)—(CH₂)_(n)—C(O)OM, each n being, independently of theothers, an integer ranging from 0 to 4, and M is hydrogen or a counterion; the illustrated —X—O—C(O)—R′ group is linked to the 6-carbon of R;and the illustrated —X—O—C(O)—R′ group hydrolyzes under biologicalconditions to yield a group of the formula —X—OH.
 2. The compound ofclaim 1 in which X is —CH₂—.
 3. The compound of claim 2 in which the Ris a residue of ingenol-3-angelate.
 4. A compound according tostructural formula (I)R—X—O—C(O)—R′  (I) including salts and hydrates thereof, wherein: R is aresidue of a phorbol ester, wherein the phorbol ester isingenol-3-acetate, ingenol-3-propionate, ingenol-3-palmitate,ingenol-3-angelate or ingenol-3-benzoate; X is an alkylene chaincontaining from 1 to 12 carbon atoms; R′ is a group of the formula —Y—Z,wherein: Y is a branched or unbranched, saturated or unsaturatedalkylene chain containing from 1 to 4 carbon atoms; Z is selected from—C(O)OM, —NR^(b)R^(b) and —NR^(c)R^(c)R^(c); M is hydrogen or a counterion; each R^(b) is, independently of the other, selected from hydrogen,lower alkyl, lower hydroxyalkyl, lower alkoxyalkyl, and heteroalkyl, or,alternatively, two R^(b) groups bonded to the same nitrogen atom may betaken together with the nitrogen atom to which they are bonded to form a5- to 7-membered heteroatomic ring; and each R^(c) is, independently ofthe others, selected from lower alkyl, lower hydroxyalkyl, loweralkoxyalkyl, and heteroalkyl; the illustrated —X—O—C(O)—R′ group islinked to the 6-carbon of R; and the illustrated —X—O—C(O)—R′ grouphydrolyzes under biological conditions to yield a group of the formula—X—OH.
 5. The compound of claim 4 in which X is —CH₂—.
 6. The compoundof claim 4 which has one or more features selected from: any alkyl groupor moiety is a branched or unbranched alkanyl; Y is an alkano; eachR^(b) is, independently of the other, selected from hydrogen and loweralkanyl; each R^(c) is, independently of the others, selected from loweralkanyl; and NR^(b)R^(b) is selected from morpholinyl, N-morpholinyl,piperazinyl, 1 piperazinyl, 1 methyl-piperazinyl and 1 methyl 4piperazinyl.
 7. The compound of claim 6 in which Y is selected frommethano, ethano, propano and butano.
 8. The compound of claim 4 in whichZ is —C(O)OM.
 9. The compound of claim 4 in which Z is selected fromdimethylamino, diethylamino, trimethylamino, triethylamino, andN-morpholinyl.
 10. The compound of claim 4 in which R is a residue ofingenol-3-angelate.
 11. A compound according to structural formula

wherein X is an alkylene chain containing from 1 to 12 carbon atoms; R′is a moiety that is ionizable at a pH in the range of about 2.0-8.0,wherein R′ comprises a carboxyl group of the formula —(CH₂)_(m)—C(O)OM,m being an integer ranging from 1 to 4, or wherein R′ comprises an aminoacid selected from —(CH₂)_(n)—CH[(CH₂)_(n)—NH₂]—(CH₂)_(n)—C(O)OM and—(CH₂)_(n)—CH(NH₂)—(CH₂)_(n)—C(O)OM, each n being, independently of theothers, an integer ranging from 0 to 4, and M is hydrogen or a counterion; R″ is selected from methyl, lower n-alkyl and branched alkenyl; andthe illustrated —X—O—C(O)—R′ group hydrolyzes under biologicalconditions to yield a group of the formula —X—OH.
 12. The compound ofclaim 11 in which X is —CH₂—.
 13. The compound of claim 12 in which R″is a branched alkenyl.
 14. A compound according to structural formula

wherein X is an alkylene chain containing from 1 to 12 carbon atoms; R′is a group of the formula —Y—Z, wherein: Y is a branched or unbranched,saturated or unsaturated alkylene chain containing from 1 to 4 carbonatoms; Z is selected from —C(O)OM, —NR^(b)R^(b) and —NR^(c)R^(c)R^(c); Mis hydrogen or a counter ion; each R^(b) is, independently of the other,selected from hydrogen, lower alkyl, lower hydroxyalkyl, loweralkoxyalkyl, and heteroalkyl, or, alternatively, two R^(b) groups bondedto the same nitrogen atom may be taken together with the nitrogen atomto which they are bonded to form a 5- to 7-membered heteroatomic ring;and each R^(c) is, independently of the others, selected from loweralkyl, lower hydroxyalkyl, lower alkoxyalkyl, and heteroalkyl; R″ isselected from methyl, lower n-alkyl and branched alkenyl; and theillustrated —X—O—C(O)—R′ group hydrolyzes under biological conditions toyield a group of the formula —X—OH.
 15. The compound of claim 14 inwhich X is —CH₂—.
 16. The compound of claim 14 which has one or morefeatures selected from: any alkyl group or moiety is a branched orunbranched alkanyl; Y is an alkano; each R^(b) is, independently of theother, selected from hydrogen and lower alkanyl; each R^(c) is,independently of the others, selected from lower alkanyl; andNR^(b)R^(b) is selected from morpholinyl, N-morpholinyl, piperazinyl, 1piperazinyl, 1 methyl-piperazinyl and 1 methyl 4 piperazinyl.
 17. Thecompound of claim 16 in which Y is selected from methano, ethano,propano and butano.
 18. The compound of claim 14 in which Z is —C(O)OM.19. The compound of claim 14 in which Z is selected from dimethylamino,diethylamino, trimethylamino, triethylamino, and N-morpholinyl.
 20. Thecompound of claim 14 in which R″ is a branched alkenyl.